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Cell_Biology_Alberts. lipases phosphatases sulfatases phospholipases CYTOSOL + H+ pump ATPADPPi H+ Figure 13–37 Lysosomes. The acid hydrolases are hydrolytic enzymes that are active under acidic conditions. an h+ aTpase in the membrane pumps h+ into the lysosome, maintaining its lumen at an acidic ph.


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Cell_Biology_Alberts. digestion of contents hydrolytically active, low-pH compartments hydrolytic enzymes. It is especially hard to apply a narrower definition than this in plant cells, as we discuss next. plant and fungal vacuoles are remarkably versatile lysosomes


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Cell_Biology_Alberts. plant and fungal vacuoles are remarkably versatile lysosomes Most plant and fungal cells (including yeasts) contain one or several very large, fluid-filled vesicles called vacuoles. They typically occupy more than 30% of the cell volume, and as much as 90% in some cell types (Figure 13–40). Vacuoles are related to animal cell lysosomes and contain a variety of hydrolytic enzymes, but their functions are remarkably diverse. The plant vacuole can act as a storage organelle for both nutrients and waste products, as a degradative compartment, as an economical way of increasing cell size, and as a controller of turgor pressure (the osmotic pressure that pushes outward on the cell wall and keeps the plant from wilting) (Figure 13–41). The same cell may have different vacuoles with distinct functions, such as digestion and storage.


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Cell_Biology_Alberts. The vacuole is important as a homeostatic device, enabling plant cells to withstand wide variations in their environment. When the pH in the environment drops, for example, the flux of H+ into the cytosol is balanced, at least in part, by an increased transport of H+ into the vacuole, which tends to keep the pH in the cytosol constant. Similarly, many plant cells maintain an almost constant turgor pressure despite large changes in the tonicity of the fluid in their immediate environment. They do so by changing the osmotic pressure of the cytosol and vacuole—in part by the controlled breakdown and resynthesis of polymers such as polyphosphate in the vacuole, and in part by altering the transport rates of sugars,


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Cell_Biology_Alberts. Figure 13–39 A model for lysosome maturation. late endosomes fuse with preexisting lysosomes (bottom arrow) or preexisting endolysosomes (top arrow). endolysosomes eventually mature into lysosomes as hydrolases complete the digestion of their contents, which can include intralumenal vesicles. lysosomes also fuse with phagosomes, as we discuss later. Figure 13–40 The plant cell vacuole.


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Cell_Biology_Alberts. Figure 13–40 The plant cell vacuole. (a) a confocal image of cells from an arabidopsis embryo that is expressing an aquaporin—yfp (yellow fluorescent protein) fusion protein in its tonoplast, or vacuole membrane (green); the cell walls have been false-colored orange. each cell contains several large vacuoles. (b) This electron micrograph of cells in a young tobacco leaf shows the cytosol as a thin layer, containing chloroplasts, pressed against the cell wall by the enormous vacuole. (a, courtesy of c. carroll and l. frigerio, based on s. gattolin et al., Mol. Plant 4:180–189, 2011. with permission from oxford university press; b, courtesy of J. burgess.) amino acids, and other metabolites across the plasma membrane and the vacuolar membrane. The turgor pressure regulates the activities of distinct transporters in each membrane to control these fluxes.


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Cell_Biology_Alberts. Humans often harvest substances stored in plant vacuoles—from rubber to opium to the flavoring of garlic. Many stored products have a metabolic function. Proteins, for example, can be preserved for years in the vacuoles of the storage cells of many seeds, such as those of peas and beans. When the seeds germinate, these proteins are hydrolyzed, and the resulting amino acids provide a food supply for the developing embryo. Anthocyanin pigments stored in vacuoles color the petals of many flowers so as to attract pollinating insects, while noxious molecules released from vacuoles when a plant is eaten or damaged provide a defense against predators. Multiple pathways deliver Materials to lysosomes Lysosomes are meeting places where several streams of intracellular traffic converge. A route that leads outward from the ER via the Golgi apparatus delivers most of the lysosome’s digestive enzymes, while at least four paths from different sources feed substances into lysosomes for digestion.


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Cell_Biology_Alberts. The best studied of these degradation paths is the one followed by macromolecules taken up from extracellular fluid by endocytosis. A similar pathway found in phagocytic cells, such as macrophages and neutrophils in vertebrates, is dedicated to the engulfment, or phagocytosis, of large particles and microorganisms to form phagosomes. A third pathway called macropinocytosis specializes in the nonspecific uptake of fluids, membrane, and particles attached to the plasma membrane. We will return to discuss these pathways later in the chapter. A fourth pathway called autophagy originates in the cytoplasm of the cell itself and is used to digest cytosol and worn-out organelles, as we discuss next. The four paths to degradation in lysosomes are illustrated in Figure 13–42.


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Cell_Biology_Alberts. Figure 13–41 The role of the vacuole in controlling the size of plant cells. a plant cell can achieve a large increase in volume without increasing the volume of the cytosol. localized weakening of the cell wall orients a turgor-driven cell enlargement that accompanies the uptake of water into an expanding vacuole. The cytosol is eventually confined to a thin peripheral layer, which is connected to the nuclear region by strands of cytosol stabilized by bundles of actin filaments (not shown). Figure 13–42 Four pathways to degradation in lysosomes. Materials in each pathway are derived from a different source. note that the autophagosome has a double membrane. In all cases, the final step is the fusion with lysosomes.


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Cell_Biology_Alberts. All cell types dispose of obsolete parts by a lysosome-dependent process called autophagy, or “self-eating.” The degradation process is important during normal cell growth and in development, where it helps restructure differentiating cells, but also in adaptive responses to stresses such as starvation and infection. Autophagy can remove large objects—macromolecules, large protein aggregates, and even whole organelles—that other disposal mechanisms such as proteasomal degradation cannot handle. Defects in autophagy may prevent cells from clearing away invading microbes, unwanted protein aggregates and abnormal proteins, and thereby contribute to diseases ranging from infectious disorders to neurodegeneration and cancer.


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Cell_Biology_Alberts. In the initial stages of autophagy, cytoplasmic cargo becomes surrounded by a double membrane that assembles by the fusion of small vesicles of unknown origin, forming an autophagosome (Figure 13–43). A few tens of different proteins have been identified in yeast and animal cells that participate in the process, which must be tightly regulated: either too little or too much can be deleterious. The whole process occurs in the following sequence of steps: 1. Induction by activation of signaling molecules: Protein kinases (including the mTOR complex 1, discussed in Chapter 15) that relay information about the metabolic status of the cell, become activated and signal to the autophagic machinery. 2.


Cell_Biology_Alberts

Cell_Biology_Alberts. 2. Nucleation and extension of a delimiting membrane into a crescent-shaped cup: Membrane vesicles, characterized by the presence of ATG9, the only transmembrane protein involved in the process, are recruited to an assembly site, where they nucleate autophagosome formation. ATG9 is not incorporated into the autophagosome: a retrieval pathway must remove it from the assembling structure. 3. Closure of the membrane cup around the target to form a sealed double-membrane-enclosed autophagosome. 4. Fusion of the autophagosome with lysosomes, catalyzed by SNAREs. 5. Digestion of the inner membrane and the lumenal contents of the autophagosome.


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Cell_Biology_Alberts. 4. Fusion of the autophagosome with lysosomes, catalyzed by SNAREs. 5. Digestion of the inner membrane and the lumenal contents of the autophagosome. Autophagy can be either nonselective or selective. In nonselective autophagy, a bulk portion of cytoplasm is sequestered in autophagosomes. It might occur, for example, in starvation conditions: when external nutrients are limiting, metabolites derived from the digestion of the captured cytosol might help the cell survive. In selective autophagy specific cargo is packaged into autophagosomes that tend to contain little cytosol, and their shape reflects the shape of the cargo. Selective autophagy mediates the degradation of worn out, or otherwise unwanted, mitochondria, peroxisomes, ribosomes, and ER; it can also be used to destroy invading microbes. Figure 13–43 A model of autophagy.


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Cell_Biology_Alberts. Figure 13–43 A model of autophagy. (a) activation of a signaling pathway initiates a nucleation event in the cytoplasm. a crescent of autophagosomal membrane grows by fusion of vesicles of unknown origin and eventually fuses to form a double-membrane-enclosed autophagosome, which sequesters a portion of the cytoplasm. The autophagosome then fuses with lysosomes containing acid hydrolases that digest its content. during the formation of the autophagosome membrane, a ubiquitinlike protein becomes activated by covalent attachment of a phosphatidylethanolamine lipid anchor. These proteins then mediate vesicle tethering and fusion, leading to the formation of a crescent-shaped membrane structure that assembles around its target (not shown). (b) an electron micrograph of an autophagosome containing a mitochondrion and a peroxisome. (b, courtesy of daniel s. friend, from d.w. fawcett, a Textbook of histology, 12th ed. new york: chapman and hall, 1994. with permission from kluwer.)


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Cell_Biology_Alberts. The selective autophagy of worn out or damaged mitochondria is called mitophagy. As discussed in Chapters 12 and 14, when mitochondria function normally, the inner mitochondrial membrane is energized by an electrochemical H+ gradient that drives ATP synthesis and the import of mitochondrial precursor proteins and metabolites. Damaged mitochondria cannot maintain the gradient, so protein import is blocked. As a consequence, a protein kinase called Pink1, which is normally imported into mitochondria, is instead retained on the mitochondrial surface where it recruits the ubiquitin ligase Parkin from the cytosol. Parkin ubiquitylates mitochondrial outer membrane proteins, which mark the organelle for selective destruction in autophagosomes. Mutations in Pink1 or Parkin cause a form of early-onset Parkinson’s disease, a degenerative disorder of the central nervous system. It is not known why the neurons that die prematurely in this disease are particularly reliant on mitophagy.


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Cell_Biology_Alberts. a Mannose 6-phosphate receptor sorts lysosomal hydrolases in the Trans golgi network We now consider the pathway that delivers lysosomal hydrolases from the TGN to lysosomes. The enzymes are first delivered to endosomes in transport vesicles that bud from the TGN, before they move on to endolysosomes and lysosomes (see Figure 13–39). The vesicles that leave the TGN incorporate the lysosomal proteins and exclude the many other proteins being packaged into different transport vesicles for delivery elsewhere.


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Cell_Biology_Alberts. How are lysosomal hydrolases recognized and selected in the TGN with the required accuracy? In animal cells they carry a unique marker in the form of mannose 6-phosphate (M6P) groups, which are added exclusively to the N-linked oligosaccharides of these soluble lysosomal enzymes as they pass through the lumen of the cis Golgi network (Figure 13–44). Transmembrane M6P receptor proteins, which are present in the TGN, recognize the M6P groups and bind to the lysosomal hydrolases on the lumenal side of the membrane and to adaptor proteins in assembling clathrin coats on the cytosolic side. In this way, the receptors help package the hydrolases into clathrin-coated vesicles that bud from the TGN and deliver their contents to early endosomes.


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Cell_Biology_Alberts. The M6P receptor protein binds to M6P at pH 6.5–6.7 in the TGN lumen and releases it at pH 6, which is the pH in the lumen of endosomes. Thus, after the receptor is delivered, the lysosomal hydrolases dissociate from the M6P receptors, which are retrieved into transport vesicles that bud from endosomes. These vesicles are coated with retromer, a coat protein complex specialized for endosome-to-TGN transport, which returns the receptors to the TGN for reuse (Figure 13–45). Transport in either direction requires signals in the cytoplasmic tail of the M6P receptor that direct this protein to the endosome or back to the TGN. These signals are recognized by the retromer complex that recruits M6P receptors into transport vesicles that bud from endosomes. The recycling of the M6P receptor resembles the recycling of the KDEL receptor discussed earlier, although it differs in the type of coated vesicles that mediate the transport.


Cell_Biology_Alberts

Cell_Biology_Alberts. Not all the hydrolase molecules that are tagged with M6P get to lysosomes. Some escape the normal packaging process in the trans Golgi network and are transported “by default” to the cell surface, where they are secreted into the extracellular fluid. Some M6P receptors, however, also take a detour to the plasma membrane, where they recapture the escaped lysosomal hydrolases and return them by receptor-mediated endocytosis (discussed later) to lysosomes via early and late endosomes. As lysosomal hydrolases require an acidic milieu to work, they can do little harm in the extracellular fluid, which usually has a neutral pH of 7.4.


Cell_Biology_Alberts

Cell_Biology_Alberts. For the sorting system that segregates lysosomal hydrolases and dispatches them to endosomes to work, the M6P groups must be added only to the appropriate glycoproteins in the Golgi apparatus. This requires specific recognition of the hydrolases by the Golgi enzymes responsible for adding M6P. Since all glycoproteins leave the ER with identical N-linked oligosaccharide chains, the signal for Figure 13–44 The structure of mannose 6-phosphate on a lysosomal hydrolase. adding the M6P units to oligosaccharides must reside somewhere in the polypeptide chain of each hydrolase. Genetic engineering experiments have revealed that the recognition signal is a cluster of neighboring amino acids on each protein’s surface, known as a signal patch (Figure 13–46). Since most lysosomal hydrolases contain multiple oligosaccharides, they acquire many M6P groups, providing a high-affinity signal for the M6P receptor. defects in the glcnac phosphotransferase cause a lysosomal storage disease in humans


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Cell_Biology_Alberts. Genetic defects that affect one or more of the lysosomal hydrolases cause a number of human lysosomal storage diseases. The defects result in an accumulation of undigested substrates in lysosomes, with severe pathological consequences, most often in the nervous system. In most cases, there is a mutation in a structural gene that codes for an individual lysosomal hydrolase. This occurs in Hurler’s disease, for example, in which the enzyme required for the breakdown of certain types of glycosaminoglycan chains is defective or missing. The most severe form of lysosomal storage disease, however, is a very rare inherited metabolic disorder called inclusion-cell disease (I-cell disease). In this condition, almost all of the hydrolytic enzymes are missing from the lysosomes of many cell types, and their undigested substrates accumulate in these lysosomes, which consequently form large inclusions in the cells. The consequent pathology is complex, affecting all organ systems, skeletal


Cell_Biology_Alberts

Cell_Biology_Alberts. types, and their undigested substrates accumulate in these lysosomes, which consequently form large inclusions in the cells. The consequent pathology is complex, affecting all organ systems, skeletal integrity, and mental development; individuals rarely live beyond six or seven years.


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Cell_Biology_Alberts. I-cell disease is due to a single gene defect and, like most genetic enzyme deficiencies, it is recessive—that is, it occurs only in individuals having two copies of the defective gene. In patients with I-cell disease, all the hydrolases missing from lysosomes are found in the blood: because they fail to sort properly in the Golgi apparatus, they are secreted rather than transported to lysosomes. The mis-sorting has been traced to a defective or missing GlcNAc phosphotransferase. Because lysosomal enzymes are not phosphorylated in the cis Golgi network, the M6P receptors do not segregate them into the appropriate transport vesicles in the TGN. Instead, the lysosomal hydrolases are carried to the cell surface and secreted. In I-cell disease, the lysosomes in some cell types, such as hepatocytes, contain a normal complement of lysosomal enzymes, implying that there is another


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Cell_Biology_Alberts. Figure 13–45 The transport of newly synthesized lysosomal hydrolases to endosomes. The sequential action of two enzymes in the cis and trans golgi network adds mannose 6-phosphate (M6p) groups to the precursors of lysosomal enzymes (see figure 13–46). The M6p-tagged hydrolases then segregate from all other types of proteins in the Tgn because adaptor proteins (not shown) in the clathrin coat bind the M6p receptors, which, in turn, bind the M6p-modified lysosomal hydrolases. The clathrin-coated vesicles bud off from the Tgn, shed their coat, and fuse with early endosomes. at the lower ph of the endosome, the hydrolases dissociate from the M6p receptors, and the empty receptors are retrieved in retromercoated vesicles to the Tgn for further rounds of transport. In the endosomes, the phosphate is removed from the M6p attached to the hydrolases, which may further ensure that the hydrolases do not return to the Tgn with the receptor.


Cell_Biology_Alberts

Cell_Biology_Alberts. with GlcNAc attached to mannose in oligosaccharide pathway for directing hydrolases to lysosomes that is used by some cell types but not others. Alternative sorting receptors function in these M6P-independent pathways. Similarly, an M6P-independent pathway in all cells sorts the membrane proteins of lysosomes from the TGN for transport to late endosomes, and those proteins are therefore normal in I-cell disease.


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Cell_Biology_Alberts. Targeting of material to lysosomes is not necessarily the end of the pathway. Lysosomal secretion of undigested content enables all cells to eliminate indigestible debris. For most cells, this seems to be a minor pathway, used only when the cells are stressed. Some cell types, however, contain specialized lysosomes that have acquired the necessary machinery for fusion with the plasma membrane. Melanocytes in the skin, for example, produce and store pigments in their lysosomes. These pigment-containing melanosomes release their pigment into the extracellular space of the epidermis by exocytosis. The pigment is then taken up by keratinocytes, leading to normal skin pigmentation. In some genetic disorders, defects in melanosome exocytosis block this transfer process, leading to forms of hypopigmentation (albinism). Under certain conditions, multivesicular bodies can also fuse with the plasma membrane. If that occurs, their intralumenal vesicles are released from cells. Circulating small


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Cell_Biology_Alberts. (albinism). Under certain conditions, multivesicular bodies can also fuse with the plasma membrane. If that occurs, their intralumenal vesicles are released from cells. Circulating small vesicles, also called exosomes, have been observed in the blood and may be used to transport components between cells, although the importance of such a mechanism of potential communication between distant cells is unknown. Some exosomes may derive from direct vesicle budding events at the plasma membrane, which is a topologically equivalent process (see Figure 13–57).


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Cell_Biology_Alberts. Lysosomes are specialized for the intracellular digestion of macromolecules. They contain unique membrane proteins and a wide variety of soluble hydrolytic enzymes that operate best at pH 5, which is the internal pH of lysosomes. An ATP-driven H+ pump in the lysosomal membrane maintains this low pH. Newly synthesized lysosomal proteins transported from the lumen of the ER, through the Golgi apparatus; they are then carried from the trans Golgi network to endosomes by means of clathrin-coated transport vesicles, before moving on to lysosomes.


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Cell_Biology_Alberts. The lysosomal hydrolases contain N-linked oligosaccharides that are covalently modified in a unique way in the cis Golgi so that their mannoses are phosphorylated. These mannose 6-phosphate (M6P) groups are recognized by an M6P receptor protein in the trans Golgi network that segregates the hydrolases and helps package them into budding transport vesicles that deliver their contents to endosomes. The M6P receptors shuttle back and forth between the trans Golgi network and the endosomes. The low pH in endosomes and the removal of the phosphate from the


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Cell_Biology_Alberts. Figure 13–46 The recognition of a lysosomal hydrolase. a glcnac phosphotransferase recognizes lysosomal hydrolases in the golgi apparatus. The enzyme has separate catalytic and recognition sites. The catalytic site binds both high-mannose n-linked oligosaccharides and udp-glcnac. The recognition site binds to a signal patch that is present only on the surface of lysosomal hydrolases. a second enzyme cleaves off the glcnac, leaving the mannose 6-phosphate exposed. M6P group cause the lysosomal hydrolases to dissociate from these receptors, making the transport of the hydrolases unidirectional. A separate transport system uses clathrin-coated vesicles to deliver resident lysosomal membrane proteins from the trans Golgi network to endosomes. TransporT InTo The cell froM The plasMa MeMbrane: endocyTosIs


Cell_Biology_Alberts

Cell_Biology_Alberts. TransporT InTo The cell froM The plasMa MeMbrane: endocyTosIs The routes that lead inward from the cell surface start with the process of endocytosis, by which cells take up plasma membrane components, fluid, solutes, macromolecules, and particulate substances. Endocytosed cargo includes receptor– ligand complexes, a spectrum of nutrients and their carriers, extracellular matrix components, cell debris, bacteria, viruses, and, in specialized cases, even other cells. Through endocytosis, the cell regulates the composition of its plasma membrane in response to changing extracellular conditions.


Cell_Biology_Alberts

Cell_Biology_Alberts. In endocytosis, the material to be ingested is progressively enclosed by a small portion of the plasma membrane, which first invaginates and then pinches off to form an endocytic vesicle containing the ingested substance or particle. Most eukaryotic cells constantly form endocytic vesicles, a process called pinocytosis (“cell drinking”); in addition, some specialized cells contain dedicated pathways that take up large particles on demand, a process called phagocytosis (“cell eating”). Endocytic vesicles form at the plasma membrane by multiple mechanisms that differ in both the molecular machinery used and how that machinery is regulated.


Cell_Biology_Alberts

Cell_Biology_Alberts. Once generated at the plasma membrane, most endocytic vesicles fuse with a common receiving compartment, the early endosome, where internalized cargo is sorted: some cargo molecules are returned to the plasma membrane, either directly or via a recycling endosome, and others are designated for degradation by inclusion in a late endosome. Late endosomes form from a bulbous, vacuolar portion of early endosomes by a process called endosome maturation. This conversion process changes the protein composition of the endosome membrane, patches of which invaginate and become incorporated within the organelles as intralumenal vesicles, while the endosome itself moves from the cell periphery to a location close to the nucleus. As an endosome matures, it ceases to recycle material to the plasma membrane and irreversibly commits its remaining contents to degradation: late endosomes fuse with one another and with lysosomes to form endolysosomes, which degrade their contents, as discussed earlier


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Cell_Biology_Alberts. and irreversibly commits its remaining contents to degradation: late endosomes fuse with one another and with lysosomes to form endolysosomes, which degrade their contents, as discussed earlier (Figure 13–47).


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Cell_Biology_Alberts. Each of the stages of endosome maturation—from the early endosome to the endolysosome—is connected through bidirectional vesicle transport pathways to


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Cell_Biology_Alberts. Figure 13–47 Endosome maturation: the endocytic pathway from the plasma membrane to lysosomes. endocytic vesicles fuse near the cell periphery with an early endosome, which is the primary sorting station. Tubular portions of the early endosome bud off vesicles that recycle endocytosed cargo back to the plasma membrane—either directly, or indirectly via recycling endosomes. recycling endosomes can store proteins until they are needed. conversion of early endosomes to late endosomes is accompanied by loss of the tubular projections. Membrane proteins destined for degradation are internalized in intralumenal vesicles. The developing late endosome, or multivesicular body, moves on microtubules to the cell interior. fully matured late endosomes no longer send vesicles to the plasma membrane, and they fuse with one another and with endolysosomes and lysosomes to degrade their contents. each stage of endosome maturation is connected via transport vesicles with the Tgn, providing a continuous


Cell_Biology_Alberts

Cell_Biology_Alberts. they fuse with one another and with endolysosomes and lysosomes to degrade their contents. each stage of endosome maturation is connected via transport vesicles with the Tgn, providing a continuous supply of newly synthesized lysosomal proteins.


Cell_Biology_Alberts

Cell_Biology_Alberts. 0.1 µm the TGN. These pathways allow insertion of newly synthesized materials, such as lysosomal enzymes arriving from the ER, and the retrieval of components, such as the M6P receptor, back into the early parts of the secretory pathway. We next discuss how the cell uses and controls the various features of endocytic trafficking. pinocytic vesicles form from coated pits in the plasma Membrane


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Cell_Biology_Alberts. Virtually all eukaryotic cells continually ingest portions of their plasma membrane in the form of small pinocytic (endocytic) vesicles. The rate at which plasma membrane is internalized in this process of pinocytosis varies between cell types, but it is usually surprisingly high. A macrophage, for example, ingests 25% of its own volume of fluid each hour. This means that it must ingest 3% of its plasma membrane each minute, or 100% in about half an hour. Fibroblasts endocytose at a somewhat lower rate (1% of their plasma membrane per minute), whereas some amoebae ingest their plasma membrane even more rapidly. Since a cell’s surface area and volume remain unchanged during this process, it is clear that the same amount of membrane being removed by endocytosis is being added to the cell surface by the converse process of exocytosis. In this sense, endocytosis and exocytosis are linked processes that can be considered to constitute an endocytic– exocytic cycle. The coupling between


Cell_Biology_Alberts

Cell_Biology_Alberts. surface by the converse process of exocytosis. In this sense, endocytosis and exocytosis are linked processes that can be considered to constitute an endocytic– exocytic cycle. The coupling between exocytosis and endocytosis is particularly strict in specialized structures characterized by high membrane turnover, such as a nerve terminal.


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Cell_Biology_Alberts. The endocytic part of the cycle often begins at clathrin-coated pits. These specialized regions typically occupy about 2% of the total plasma membrane area. The lifetime of a clathrin-coated pit is short: within a minute or so of being formed, it invaginates into the cell and pinches off to form a clathrin-coated vesicle (Figure 13–48). About 2500 clathrin-coated vesicles pinch off from the plasma membrane of a cultured fibroblast every minute. The coated vesicles are even more transient than the coated pits: within seconds of being formed, they shed their coat and fuse with early endosomes. not all pinocytic vesicles are clathrin-coated


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Cell_Biology_Alberts. not all pinocytic vesicles are clathrin-coated In addition to clathrin-coated pits and vesicles, cells can form other types of pinocytic vesicles, such as caveolae (from the Latin for “little cavities”), originally recognized by their ability to transport molecules across endothelial cells that form the inner lining of blood vessels. Caveolae, sometimes seen in the electron microscope as deeply invaginated flasks, are present in the plasma membrane of most vertebrate cell types (Figure 13–49). They are thought to form from lipid rafts in the plasma membrane (discussed in Chapter 10), which are especially rich


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Cell_Biology_Alberts. Figure 13–48 The formation of clathrincoated vesicles from the plasma membrane. These electron micrographs illustrate the probable sequence of events in the formation of a clathrin-coated vesicle from a clathrin-coated pit. The clathrincoated pits and vesicles shown are larger than those seen in normal-sized cells; they are from a very large hen oocyte and they take up lipoprotein particles to form yolk. The lipoprotein particles bound to their membrane-bound receptors appear as a dense, fuzzy layer on the extracellular surface of the plasma membrane—which is the inside surface of the coated pit and vesicle. (courtesy of M.M. perry and a.b. gilbert, J. Cell sci. 39:257–272, 1979. with permission from The company of biologists.) 0.2 µm in cholesterol, glycosphingolipids, and glycosylphosphatidylinositol (GPI)-anchored membrane proteins (see Figure 10–13). The major structural proteins in caveolae are caveolins, a family of unusual integral membrane proteins that each insert a


Cell_Biology_Alberts

Cell_Biology_Alberts. (GPI)-anchored membrane proteins (see Figure 10–13). The major structural proteins in caveolae are caveolins, a family of unusual integral membrane proteins that each insert a hydrophobic loop into the membrane from the cytosolic side but do not extend across the membrane. On their cytosolic side, caveolins are bound to large protein complexes of caving proteins, which are thought to stabilize the membrane curvature.


Cell_Biology_Alberts

Cell_Biology_Alberts. In contrast to clathrin-coated and COPIor COPII-coated vesicles, caveolae are usually static structures. Nonetheless, they can be induced to pinch off and serve as endocytic vesicles to transport cargo to early endosomes or to the plasma membrane on the opposite side of a polarized cell (in a process called transcytosis, which we discuss later). Some animal viruses such as SV40 and papillomavirus (which causes warts) enter cells in vesicles derived from caveolae. The viruses are first delivered to early endosomes and move from there in transport vesicles to the lumen of the ER. The viral genome exits across the ER membrane into the cytosol, from where it is imported into the nucleus to start the infection cycle. Cholera toxin (discussed in Chapters 15 and 19) also enters the cell through caveolae and is transported to the ER before entering the cytosol.


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Cell_Biology_Alberts. Macropinocytosis is another clathrin-independent endocytic mechanism that can be activated in practically all animal cells. In most cell types, it does not operate continually but rather is induced for a limited time in response to cell-surface receptor activation by specific cargoes, including growth factors, integrin ligands, apoptotic cell remnants, and some viruses. These ligands activate a complex signaling pathway, resulting in a change in actin dynamics and the formation of cell-surface protrusions, called ruffles (discussed in Chapter 16). When ruffles collapse back onto the cell, large fluid-filled endocytic vesicles form, called macropinosomes (Figure 13–50), which transiently increase the bulk fluid uptake of a cell by up to tenfold. Macropinocytosis is a dedicated degradative pathway: macropinosomes acidify and then fuse with late endosomes or endolysosomes, without recycling their cargo back to the plasma membrane.


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Cell_Biology_Alberts. cells use receptor-Mediated endocytosis to Import selected extracellular Macromolecules In most animal cells, clathrin-coated pits and vesicles provide an efficient pathway for taking up specific macromolecules from the extracellular fluid. In this process, called receptor-mediated endocytosis, the macromolecules bind to


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Cell_Biology_Alberts. Figure 13–49 Caveolae in the plasma membrane of a fibroblast. (a) This electron micrograph shows a plasma membrane with a very high density of caveolae. (b) This rapid-freeze deep-etch image demonstrates the characteristic “cauliflower” texture of the cytosolic face of the caveolae membrane. The characteristic texture is thought to result from aggregates of caveolins and cavins. a clathrin-coated pit is also seen at the upper right. (courtesy of r.g.w. anderson, from k.g. rothberg et al., Cell 68:673–682, 1992. with permission from elsevier.) complementary transmembrane receptor proteins, which accumulate in coated pits, and then enter the cell as receptor–macromolecule complexes in clathrin-coated vesicles (see Figure 13–48). Because ligands are selectively captured by receptors, receptor-mediated endocytosis provides a selective concentrating mechanism that increases the efficiency of internalization of particular ligands more than a hundredfold. In this way, even minor components


Cell_Biology_Alberts

Cell_Biology_Alberts. endocytosis provides a selective concentrating mechanism that increases the efficiency of internalization of particular ligands more than a hundredfold. In this way, even minor components of the extracellular fluid can be efficiently taken up in large amounts. A particularly well-understood and physiologically important example is the process that mammalian cells use to import cholesterol.


Cell_Biology_Alberts

Cell_Biology_Alberts. Many animal cells take up cholesterol through receptor-mediated endocytosis and, in this way, acquire most of the cholesterol they require to make new membrane. If the uptake is blocked, cholesterol accumulates in the blood and can contribute to the formation in blood vessel (artery) walls of atherosclerotic plaques, deposits of lipid and fibrous tissue that can cause strokes and heart attacks by blocking arterial blood flow. In fact, it was a study of humans with a strong genetic predisposition for atherosclerosis that first revealed the mechanism of receptor-mediated endocytosis.


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Cell_Biology_Alberts. Most cholesterol is transported in the blood as cholesteryl esters in the form of lipid–protein particles known as low-density lipoproteins (LDLs) (Figure 13–51). When a cell needs cholesterol for membrane synthesis, it makes transmembrane receptor proteins for LDL and inserts them into its plasma membrane. Once in the plasma membrane, the LDL receptors diffuse until they associate with clathrin-coated pits that are in the process of forming. There, an endocytosis signal in the cytoplasmic tail of the LDL receptors binds the membrane-bound adaptor protein AP2 after its conformation has been locally unlocked by binding to PI(4,5)P2 on the plasma membrane. Coincidence detection, as discussed earlier, thus imparts both efficiency and selectivity to the process (see Figure 13–9). AP2 then recruits clathrin to initiate endocytosis.


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Cell_Biology_Alberts. Since coated pits constantly pinch off to form coated vesicles, any LDL particles bound to LDL receptors in the coated pits are rapidly internalized in coated vesicles. After shedding their clathrin coats, the vesicles deliver their contents to early endosomes. Once the LDL and LDL receptors encounter the low pH in early endosomes, LDL is released from its receptor and is delivered via late endosomes to lysosomes. There, the cholesteryl esters in the LDL particles are hydrolyzed to free cholesterol, which is now available to the cell for new membrane synthesis (Movie 13.3). If too much free cholesterol accumulates in a cell, the cell shuts off both its own cholesterol synthesis and the synthesis of LDL receptors, so that it ceases both to make or to take up cholesterol.


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Cell_Biology_Alberts. This regulated pathway for cholesterol uptake is disrupted in individuals who inherit defective genes encoding LDL receptors. The resulting high levels of blood cholesterol predispose these individuals to develop atherosclerosis prematurely, and many would die at an early age of heart attacks resulting from coronary artery disease if they were not treated with drugs such as statins that lower the level of blood cholesterol. In some cases, the receptor is lacking altogether. In others, the receptors are defective—in either the extracellular binding site for LDL or the Figure 13–50 Schematic representation of macropinocytosis. cell signaling events lead to a reprogramming of actin dynamics, which in turn triggers the formation of cell-surface ruffles. as the ruffles collapse back onto the cell surface, they nonspecifically trap extracellular fluid and macromolecules and particles contained in it, forming large vacuoles, or macropinosomes, as shown.


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Cell_Biology_Alberts. Figure 13–51 A low-density lipoprotein (LDL) particle. each spherical particle has a mass of 3 × 106 daltons. It contains a core of about 1500 cholesterol molecules esterified to long-chain fatty acids. a lipid monolayer composed of about 800 phospholipid and 500 unesterified cholesterol molecules surrounds the core of cholesteryl esters. a single molecule of apolipoprotein b, a 500,000-dalton beltlike protein, organizes the particle and mediates the specific binding of ldl to cell-surface ldl receptors. intracellular binding site that attaches the receptor AP2 adaptor protein in clathrin-coated pits. In the latter case, normal numbers of LDL receptors are present, but they fail to become localized in clathrin-coated pits. Although LDL binds to the surface of these mutant cells, it is not internalized, directly demonstrating the importance of clathrin-coated pits for the receptor-mediated endocytosis of cholesterol.


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Cell_Biology_Alberts. More than 25 distinct receptors are known to participate in receptor-mediated endocytosis of different types of molecules. They all apparently use clathrin-dependent internalization routes and are guided into clathrin-coated pits by signals in their cytoplasmic tails that bind to adaptor proteins in the clathrin coat. Many of these receptors, like the LDL receptor, enter coated pits irrespective of whether they have bound their specific ligands. Others enter preferentially when bound to a specific ligand, suggesting that a ligand-induced conformational change is required for them to activate the signal sequence that guides them into the pits. Since most plasma membrane proteins fail to become concentrated in clathrin-coated pits, the pits serve as molecular filters, preferentially collecting certain plasma membrane proteins (receptors) over others.


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Cell_Biology_Alberts. Electron-microscope studies of cultured cells exposed simultaneously to different labeled ligands demonstrate that many kinds of receptors can cluster in the same coated pit, whereas some other receptors cluster in different clathrin-coated pits. The plasma membrane of one clathrin-coated pit can accommodate more than 100 receptors of assorted varieties. specific proteins are retrieved from early endosomes and returned to the plasma Membrane


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Cell_Biology_Alberts. specific proteins are retrieved from early endosomes and returned to the plasma Membrane Early endosomes are the main sorting station in the endocytic pathway, just as the cis and trans Golgi networks serve this function in the secretory pathway. In the mildly acidic environment of the early endosome, many internalized receptor proteins change their conformation and release their ligand, as already discussed for the M6P receptors. Those endocytosed ligands that dissociate from their receptors in the early endosome are usually doomed to destruction in lysosomes (although cholesterol is an exception, as just discussed), along with the other soluble contents of the endosome. Some other endocytosed ligands, however, remain bound to their receptors, and thereby share the fate of the receptors.


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Cell_Biology_Alberts. In the early endosome, the LDL receptor dissociates from its ligand, LDL, and is recycled back to the plasma membrane for reuse, leaving the discharged LDL to be carried to lysosomes (Figure 13–52). The recycling transport vesicles bud from long, narrow tubules that extend from the early endosomes. It is likely that the geometry of these tubules helps the sorting process: because tubules have a large membrane area enclosing a small volume, membrane proteins become enriched over soluble proteins. The transport vesicles return the LDL receptor directly to the plasma membrane. The transferrin receptor follows a similar recycling pathway as the LDL receptor, but unlike the LDL receptor it also recycles its ligand. Transferrin is a soluble


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Cell_Biology_Alberts. The transferrin receptor follows a similar recycling pathway as the LDL receptor, but unlike the LDL receptor it also recycles its ligand. Transferrin is a soluble Figure 13–52 The receptor-mediated endocytosis of LDL. note that the ldl dissociates from its receptors in the acidic environment of the early endosome. after a number of steps, the ldl ends up in endolysosomes and lysosomes, where it is degraded to release free cholesterol. In contrast, the ldl receptors are returned to the plasma membrane via transport vesicles that bud off from the tubular region of the early endosome, as shown. for simplicity, only one ldl receptor is shown entering the cell and returning to the plasma membrane. whether it is occupied or not, an ldl receptor typically makes one round trip into the cell and back to the plasma membrane every 10 minutes, making a total of several hundred trips in its 20-hour life-span.


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Cell_Biology_Alberts. protein that carries iron in the blood. Cell-surface transferrin receptors deliver transferrin with its bound iron to early endosomes by receptor-mediated endocytosis. The low pH in the endosome induces transferrin to release its bound iron, but the iron-free transferrin itself (called apotransferrin) remains bound to its receptor. The receptor–apotransferrin complex enters the tubular extensions of the early endosome and from there is recycled back to the plasma membrane. When the apotransferrin returns to the neutral pH of the extracellular fluid, it dissociates from the receptor and is thereby freed to pick up more iron and begin the cycle again. Thus, transferrin shuttles back and forth between the extracellular fluid and early endosomes, avoiding lysosomes and delivering iron to the cell interior, as needed for cells to grow and proliferate. plasma Membrane signaling receptors are down-regulated by degradation in lysosomes


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Cell_Biology_Alberts. plasma Membrane signaling receptors are down-regulated by degradation in lysosomes A second pathway that endocytosed receptors can follow from endosomes is taken by many signaling receptors, including opioid receptors and the receptor that binds epidermal growth factor (EGF). EGF is a small, extracellular signal protein that stimulates epidermal and various other cells to divide. Unlike LDL receptors, EGF receptors accumulate in clathrin-coated pits only after binding their ligand, and most do not recycle but are degraded in lysosomes, along with the ingested EGF. EGF binding therefore first activates intracellular signaling pathways and then leads to a decrease in the concentration of EGF receptors on the cell surface, a process called receptor downregulation, that reduces the cell’s subsequent sensitivity to EGF (see Figure 15–20).


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Cell_Biology_Alberts. Receptor downregulation is highly regulated. The activated receptors are first covalently modified on the cytosolic face with the small protein ubiquitin. Unlike polyubiquitylation, which adds a chain of ubiquitins that typically targets a protein for degradation in proteasomes (discussed in Chapter 6), ubiquitin tagging for sorting into the clathrin-dependent endocytic pathway adds just one or a few single ubiquitin molecules to the protein—a process called monoubiquitylation or multiubiquitylation, respectively. Ubiquitin-binding proteins recognize the attached ubiquitin and help direct the modified receptors into clathrin-coated pits. After delivery to the early endosome, other ubiquitin-binding proteins that are part of ESCRT complexes (ESCRT = Endosome Sorting Complex Required for Transport) recognize and sort the ubiquitylated receptors into intralumenal vesicles, which are retained in the maturing late endosome (see Figure 13–47). In this way, addition of ubiquitin blocks


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Cell_Biology_Alberts. Transport) recognize and sort the ubiquitylated receptors into intralumenal vesicles, which are retained in the maturing late endosome (see Figure 13–47). In this way, addition of ubiquitin blocks receptor recycling to the plasma membrane and directs the receptors into the degradation pathway, as we discuss next.


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Cell_Biology_Alberts. The endosomal compartments can be made visible in the electron microscope by adding a readily detectable tracer molecule, such as the enzyme peroxidase, to the extracellular medium and allowing varying lengths of time for the cell to endocytose the tracer. The distribution of the molecule after its uptake reveals the sequence of events. Within a minute or so after adding the tracer, it starts to appear in early endosomes, just beneath the plasma membrane (Figure 13–53). By 5–15 minutes later, it has moved to late endosomes, close to the Golgi apparatus and near the nucleus.


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Cell_Biology_Alberts. How early endosomes arise is not entirely clear, but their membrane and volume are mainly derived from incoming endocytic vesicles that fuse with one another (Movie 13.4). Early endosomes are relatively small and patrol the cytoplasm underlying the plasma membrane in jerky back-and-forth movements along microtubules, capturing incoming vesicles. Typically, an early endosome receives incoming vesicles for about 10 minutes, during which time membrane and fluid is rapidly recycled to the plasma membrane. Some of the incoming cargo, however, accumulates over the lifetime of the early endosome, eventually to be included in the late endosome. 0.5 µm


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Cell_Biology_Alberts. 0.5 µm Figure 13–53 Electron micrograph of an early endosome. The endosome is labeled with endocytosed horseradish peroxidase, a widely used enzyme marker, detected in this case by an electron-dense reaction product. Many tubular extensions protrude from the central vacuolar space of the early endosome, which will later mature to give rise to a late endosome. (from J. Tooze and M. hollinshead, J. Cell Biol. 118:813–830, 1992.)


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Cell_Biology_Alberts. J. Tooze and M. hollinshead, J. Cell Biol. 118:813–830, 1992.) Early endosomes have tubular and vacuolar domains (see Figure 13–53). Most of the membrane surface is in the tubules and most of the volume is in the vacuolar domain. During endosome maturation, the two domains have different fates: the vacuolar portions of the early endosome are retained and transformed into late endosomes; the tubular portions shrink. Maturing endosomes, also called multivesicular bodies, migrate along microtubules toward the cell interior, shedding membrane tubules and vesicles that recycle material to the plasma membrane and TGN, and receiving newly synthesized lysosomal proteins. As they concentrate in a perinuclear region of the cell, the multivesicular bodies fuse with each other, and eventually with endolysosomes and lysosomes (see Figure 13–47).


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Cell_Biology_Alberts. Many changes occur during the maturation process. (1) The endosome changes shape and location, as the tubular domains are lost and the vacuolar domains are thoroughly modified. (2) Rab proteins, phosphoinositide lipids, fusion machinery (SNAREs and tethers), and microtubule motor proteins all participate in a molecular makeover of the cytosolic face of the endosome membrane, changing the functional characteristics of the organelle. (3) A V-type ATPase in the endosome membrane pumps H+ from the cytosol into the endosome lumen and acidifies the organelle. Crucially, the increasing acidity that accompanies maturation renders lysosomal hydrolases increasingly more active, influencing many receptor–ligand interactions, thereby controlling receptor loading and unloading. (4) Intralumenal vesicles sequester endocytosed signaling receptors inside the endosome, thus halting the receptor signaling activity. (5) Lysosome proteins are delivered from the TGN to the maturing endosome. Most of these


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Cell_Biology_Alberts. sequester endocytosed signaling receptors inside the endosome, thus halting the receptor signaling activity. (5) Lysosome proteins are delivered from the TGN to the maturing endosome. Most of these events occur gradually but eventually lead to a complete transformation of the endosome into an early endolysosome.


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Cell_Biology_Alberts. In addition to committing selected cargo for degradation, the maturation process is important for lysosome maintenance. The continual delivery of lysosome components from the TGN to maturing endosomes, ensures a steady supply of new lysosome proteins. The endocytosed materials mix in early endosomes with newly arrived acid hydrolases. Although mild digestion may start here, many hydrolases are synthesized and delivered as proenzymes, called zymogens, which contain extra inhibitory domains that keep the hydrolases inactive until these domains are proteolytically removed at later stages of endosome maturation. Moreover, the pH in early endosomes is not low enough to activate lysosomal hydrolases optimally. By these means, cells can retrieve membrane proteins intact from early endosomes and recycle them back to the plasma membrane. EsCrT protein complexes Mediate the formation of Intralumenal vesicles in Multivesicular bodies


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Cell_Biology_Alberts. EsCrT protein complexes Mediate the formation of Intralumenal vesicles in Multivesicular bodies As endosomes mature, patches of their membrane invaginate into the endosome lumen and pinch off to form intralumenal vesicles. Because of their appearance in the electron microscope such maturing endosomes are also called multivesicular bodies (Figure 13–54).


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Cell_Biology_Alberts. The multivesicular bodies carry endocytosed membrane proteins that are to be degraded. As part of the protein-sorting process, receptors destined for degradation, such as the occupied EGF receptors described previously, selectively partition into the invaginating membrane of the multivesicular bodies. In this way, both the receptors and any signaling proteins strongly bound to them are sequestered away from the cytosol where they might otherwise continue signaling. They also are made fully accessible to the digestive enzymes that eventually will degrade them (Figure 13–55). In addition to endocytosed membrane proteins, multivesicular bodies include the soluble content of early endosomes destined for late endosomes and digestion in lysosomes.


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Cell_Biology_Alberts. As discussed earlier, sorting into intralumenal vesicles requires one or multiple ubiquitin tags, which are added to the cytosolic domains of membrane proteins. These tags initially help guide the proteins into clathrin-coated vesicles in the plasma membrane. Once delivered to the endosomal membrane, the ubiquitin tags are recognized again, this time by a series of cytosolic ESCRT protein 0.5 µm


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Cell_Biology_Alberts. Figure 13–54 Electron micrograph of a multivesicular body. The large amount of internal membrane will be delivered to the lysosome, for digestion. (courtesy of andrew staehelin, from a. driouich, a. Jauneau and l.a. staehelin; Plant Physiol. 113:487–492, 1997. with permission from the american society of plant biologists.) complexes, (ESCRT-0, -I, -II, and -III), which bind sequentially and ultimately mediate the sorting process into the intralumenal vesicles. Membrane invagination into multivesicular bodies also depends on a lipid kinase that phosphorylates phosphatidylinositol to produce PI(3)P, which serves as an additional docking site for the ESCRT complexes; these complexes require both PI(3)P and the presence of ubiquitylated cargo proteins to bind to the endosomal membrane. ESCRT-III forms large multimeric assemblies on the membrane that bend the membrane (Figure 13–56).


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Cell_Biology_Alberts. Mutant cells compromised in ESCRT function display signaling defects. In such cells, activated receptors cannot be down-regulated by endocytosis and packaging into multivesicular bodies. The still-active receptors therefore mediate prolonged signaling, which can lead to uncontrolled cell proliferation and cancer. Processes that shape membranes often use similar machinery. Because of strong similarities in their protein sequences, researchers think that ESCRT complexes are evolutionarily related to components that mediate cell-membrane deformation in cytokinesis in archaea. Similarly, the ESCRT machinery that drives the internal budding from the endosome membrane to form intralumenal vesicles is also used in animal cell cytokinesis and virus budding, which are topologically equivalent, as both processes involve budding away from the cytosolic surface of the membrane (Figure 13–57).


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Cell_Biology_Alberts. The fates of endocytosed receptors—and of any ligands remaining bound to them—vary according to the specific type of receptor. As we discussed, most receptors are recycled and returned to the same plasma membrane domain from which they came; some proceed to a different domain of the plasma membrane, thereby mediating transcytosis; and some progress to lysosomes, where they are degraded. Receptors on the surface of polarized epithelial cells can transfer specific macromolecules from one extracellular space to another by transcytosis. A newborn, for example, obtains antibodies from its mother’s milk (which help protect it against infection) by transporting them across the epithelium of its gut. The lumen of the gut is acidic, and, at this low pH, the antibodies in the milk bind to specific receptors on the apical (absorptive) surface of the gut epithelial cells. The receptor–antibody complexes are internalized via clathrin-coated pits and


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Cell_Biology_Alberts. Figure 13–55 The sequestration of endocytosed proteins into intralumenal vesicles of multivesicular bodies. ubiquitylated membrane proteins are sorted into domains on the endosome membrane, which invaginate and pinch off to form intralumenal vesicles. The ubiquitin marker is removed and returned to the cytosol for reuse before the intralumenal vesicle closes. eventually, proteases and lipases in lysosomes digest all of the internal membranes. The invagination processes are essential for complete digestion of endocytosed membrane proteins: because the outer membrane of the multivesicular body becomes continuous with the lysosomal membrane, which is resistant to lysosomal hydrolases; the hydrolases, for example, could not digest the cytosolic domains of endocytosed transmembrane proteins, such as the egf receptor shown here, if the protein were not localized in intralumenal vesicles.


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Cell_Biology_Alberts. Figure 13–56 Sorting of endocytosed membrane proteins into the intralumenal vesicles of a multivesicular body. a series of complex binding events passes the ubiquitylated cargo proteins sequentially from one escrT complex (escrT-0) to the next, eventually concentrating them in membrane areas that bud into the lumen of the endosome to form intralumenal vesicles. escrT-III assembles into expansive multimeric structures and mediates invagination. The mechanisms of how cargo molecules are shepherded into the vesicles and how the vesicles are formed without including the escrT complexes themselves remain unknown. escrT complexes are soluble in the cytosol, are recruited to the membrane sequentially as needed, and are then released back into the cytosol as the vesicle pinches off.


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Cell_Biology_Alberts. vesicles and are delivered to early endosomes. The complexes remain intact and are retrieved in transport vesicles that bud from the early endosome and subsequently fuse with the basolateral domain of the plasma membrane. On exposure to the neutral pH of the extracellular fluid that bathes the basolateral surface of the cells, the antibodies dissociate from their receptors and eventually enter the baby’s bloodstream. The transcytotic pathway from the early endosome back to the plasma membrane is not direct. The receptors first move from the early endosome to the recycling endosome. The variety of pathways that different receptors follow from early endosomes implies that, in addition to binding sites for their ligands and binding sites for coated pits, many receptors also possess sorting signals that guide them into the appropriate transport pathway (Figure 13–58).


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Cell_Biology_Alberts. Cells can regulate the release of membrane proteins from recycling endosomes, thus adjusting the flux of proteins through the transcytotic pathway according to need. This regulation, the mechanism of which is uncertain, allows recycling endosomes to play an important part in adjusting the concentration of specific plasma membrane proteins. Fat cells and muscle cells, for example, contain large intracellular pools of the glucose transporters that are responsible for the uptake of glucose across the plasma membrane. These membrane transport proteins are stored in specialized recycling endosomes until the hormone insulin stimulates the cell to increase its rate of glucose uptake. In response to the insulin signal, transport vesicles rapidly bud from the recycling endosome and deliver large numbers of glucose transporters to the plasma membrane, thereby greatly increasing the rate of glucose uptake into the cell (Figure 13–59). Similarly, kidney cells regulate the insertion of aquaporins


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Cell_Biology_Alberts. of glucose transporters to the plasma membrane, thereby greatly increasing the rate of glucose uptake into the cell (Figure 13–59). Similarly, kidney cells regulate the insertion of aquaporins and V-ATPase into the plasma membrane to increase water and acid excretion, respectively, both in response to hormones.


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Cell_Biology_Alberts. Phagocytosis is a special form of endocytosis in which a cell uses large endocytic vesicles called phagosomes to ingest large particles such as microorganisms and dead cells. Phagocytosis is distinct, both in purpose and mechanism, from macropinocytosis, which we discussed earlier. In protozoa, phagocytosis is a form of feeding: large particles taken up into phagosomes end up in lysosomes, and the products of the subsequent digestive processes pass into the cytosol to be used as food. However, few cells in multicellular organisms are able to ingest such large particles efficiently. In the gut of animals, for example, extracellular processes break down food particles, and cells import the small products of hydrolysis. Figure 13–57 Conserved mechanism in multivesicular body formation and virus budding. In the two topologically equivalent processes indicated by the arrows, escrT complexes (not shown) shape membranes into buds that bulge away from the cytosol.


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Cell_Biology_Alberts. Figure 13–58 Possible fates for transmembrane receptor proteins that have been endocytosed. Three pathways from the early endosomal compartment in an epithelial cell are shown. retrieved receptors are returned (1) to the same plasma membrane domain from which they came (recycling) or (2) via a recycling endosome to a different domain of the plasma membrane (transcytosis). (3) receptors that are not specifically retrieved from early or recycling endosomes follow the pathway from the endosomal compartment to lysosomes, where they are degraded (degradation). If the ligand that is endocytosed with its receptor stays bound to the receptor in the acidic environment of the endosome, it shares the same fate as the receptor; otherwise, it is delivered to lysosomes. recycling endosomes are a way-station on the transcytotic pathway. In the transcytosis example shown here, an antibody fc receptor on a gut epithelial cell binds antibody and is endocytosed, eventually carrying the antibody to the


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Cell_Biology_Alberts. on the transcytotic pathway. In the transcytosis example shown here, an antibody fc receptor on a gut epithelial cell binds antibody and is endocytosed, eventually carrying the antibody to the basolateral plasma membrane. The receptor is called an fc receptor because it binds the fc part of the antibody (discussed in chapter 24).


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Cell_Biology_Alberts. Figure 13–59 Storage of plasma membrane proteins in recycling endosomes. recycling endosomes can serve as an intracellular storage site for specialized plasma membrane proteins that can be mobilized when needed. In the example shown, insulin binding to the insulin receptor triggers an intracellular signaling pathway that causes the rapid insertion of glucose transporters into the plasma membrane of a fat or muscle cell, greatly increasing its glucose intake.


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Cell_Biology_Alberts. Phagocytosis is important in most animals for purposes other than nutrition, and it is carried out mainly by specialized cells—so-called professional phagocytes. In mammals, two important classes of white blood cells that act as professional phagocytes are macrophages and neutrophils (Movie 13.5). These cells develop from hemopoietic stem cells (discussed in Chapter 22), and they ingest invading microorganisms to defend us against infection. Macrophages also have an important role in scavenging senescent cells and cells that have died by apoptosis (discussed in Chapter 18). In quantitative terms, the clearance of senescent and dead cells is by far the most important: our macrophages, for example, phagocytose more than 1011 senescent red blood cells in each of us every day.


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Cell_Biology_Alberts. The diameter of a phagosome is determined by the size of its ingested particles, and those particles can be almost as large as the phagocytic cell itself (Figure 13–60). Phagosomes fuse with lysosomes, and the ingested material is then degraded. Indigestible substances remain in the lysosomes, forming residual bodies that can be excreted from cells by exocytosis, as mentioned earlier. Some of the internalized plasma membrane components never reach the lysosome, because they are retrieved from the phagosome in transport vesicles and returned to the plasma membrane.


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Cell_Biology_Alberts. Some pathogenic bacteria have evolved elaborate mechanisms to prevent phagosome–lysosome fusion. The bacterium Legionella pneumophila, for example, which causes Legionnaires’ disease (discussed in Chapter 23), injects into its unfortunate host a Rab-modifying enzyme that causes certain Rab proteins to misdirect membrane traffic, thereby preventing phagosome–lysosome fusion. The bacterium, thus spared from lysosomal degradation, remains in the modified phagosome, growing and dividing as an intracellular pathogen, protected from the host’s adaptive immune system.


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Cell_Biology_Alberts. Phagocytosis is a cargo-triggered process. That is, it requires the activation of cell-surface receptors that transmit signals to the cell interior. Thus, to be phagocytosed, particles must first bind to the surface of the phagocyte (although not all particles that bind are ingested). Phagocytes have a variety of cell surface receptors that are functionally linked to the phagocytic machinery of the cell. The best-characterized triggers of phagocytosis are antibodies, which protect us by binding to the surface of infectious microorganisms (pathogens) and initiating a series of events that culminate in the invader being phagocytosed. When antibodies initially attack a pathogen, they coat it with antibody molecules that bind to Fc receptors on the surface of macrophages and neutrophils, activating the receptors to induce the phagocytic cell to extend pseudopods, which engulf the particle and fuse at their tips to form a phagosome (Figure 13–61A). Localized actin polymerization, initiated


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Cell_Biology_Alberts. the receptors to induce the phagocytic cell to extend pseudopods, which engulf the particle and fuse at their tips to form a phagosome (Figure 13–61A). Localized actin polymerization, initiated by Rho family GTPases and their activating Rho-GEFs (discussed in Chapters 15 and 16), shapes the pseudopods. The activated Rho GTPases switch on the kinase activity of local PI kinases to produce PI(4,5)P2 in the membrane (see Figure 13–11), which stimulates actin polymerization. To seal off the phagosome and complete the engulfment, actin is depolymerized by a PI 3-kinase that converts the PI(4,5)P2 to PI(3,4,5)P3, which is required for closure


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Cell_Biology_Alberts. Figure 13–60 Phagocytosis by a macrophage. a scanning electron micrograph of a mouse macrophage phagocytosing two chemically altered red blood cells. The red arrows point to edges of thin processes (pseudopods) of the macrophage that are extending as collars to engulf the red cells. (courtesy of Jean paul revel.) PI(4,5)P2 PI(3,4,5)P3 of the phagosome and may also contribute to reshaping the actin network to help drive the invagination of the forming phagosome (Figure 13–61B). In this way, the ordered generation and consumption of specific phosphoinositides guides sequential steps in phagosome formation.


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Cell_Biology_Alberts. Several other classes of receptors that promote phagocytosis have been characterized. Some recognize complement components, which collaborate with antibodies in targeting microbes for destruction (discussed in Chapter 24). Others directly recognize oligosaccharides on the surface of certain pathogens. Still others recognize cells that have died by apoptosis. Apoptotic cells lose the asymmetric distribution of phospholipids in their plasma membrane. As a consequence, negatively charged phosphatidylserine, which is normally confined to the cytosolic leaflet of the lipid bilayer, is now exposed on the outside of the cell, where it helps to trigger the phagocytosis of the dead cell.


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Cell_Biology_Alberts. Remarkably, macrophages will also phagocytose a variety of inanimate particles—such as glass or latex beads and asbestos fibers—yet they do not phagocytose live cells in their own body. The living cells display “don’t-eat-me” signals in the form of cell-surface proteins that bind to inhibiting receptors on the surface of macrophages. The inhibitory receptors recruit tyrosine phosphatases that antagonize the intracellular signaling events required to initiate phagocytosis, thereby locally inhibiting the phagocytic process. Thus phagocytosis, like many other cell processes, depends on a balance between positive signals that activate the process and negative signals that inhibit it. Apoptotic cells are thought both to gain “eat-me” signals (such as extracellularly exposed phosphatidylserine) and to lose their “don’t-eat-me” signals, causing them to be very rapidly phagocytosed by macrophages.


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Cell_Biology_Alberts. Cells ingest fluid, molecules, and particles by endocytosis, in which localized regions of the plasma membrane invaginate and pinch off to form endocytic vesicles. In most cells, endocytosis internalizes a large fraction of the plasma membrane every hour. The cells remain the same size because most of the plasma membrane components (proteins and lipids) that are endocytosed are continually returned to the cell surface by exocytosis. This large-scale endocytic–exocytic cycle is mediated largely by clathrin-coated pits and vesicles but clathrin-independent endocytic pathways also contribute. While many of the endocytosed molecules are quickly recycled to the plasma membrane, others eventually end up in lysosomes, where they are degraded. Most of the ligands that are endocytosed with their receptors dissociate from their receptors


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Cell_Biology_Alberts. Figure 13–61 A neutrophil reshaping its plasma membrane during phagocytosis. (a) an electron micrograph of a neutrophil phagocytosing a bacterium, which is in the process of dividing. (b) pseudopod extension and phagosome formation are driven by actin polymerization and reorganization, which respond to the accumulation of specific phosphoinositides in the membrane of the forming phagosome: pI(4,5)p2 stimulates actin polymerization, which promotes pseudopod formation, and then pI(3,4,5)p3 depolymerizes actin filaments at the base. (a, courtesy of dorothy f. bainton, phagocytic Mechanisms in health and disease. new york: Intercontinental Medical book corporation, 1971.) in the acidic environment of the endosome and eventually end up in lysosomes, while most of the receptors are recycled via transport vesicles back to the cell surface for reuse. Many cell-surface signaling receptors become tagged with ubiquitin when activated by binding their extracellular ligands. Ubiquitylation guides


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Cell_Biology_Alberts. transport vesicles back to the cell surface for reuse. Many cell-surface signaling receptors become tagged with ubiquitin when activated by binding their extracellular ligands. Ubiquitylation guides activated receptors into clathrin-coated pits, they and their ligands are efficiently internalized and delivered to early endosomes.


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Cell_Biology_Alberts. Early endosomes, rapidly mature into late endosomes. During maturation, patches of the endosomal membrane containing ubiquitylated receptors invaginate and pinch off to form intralumenal vesicles. This process is mediated by ESCRT complexes and sequesters the receptors away from the cytosol, which terminates their signaling activity. Late endosomes migrate along microtubules toward the interior of the cell where they fuse with one another and with lysosomes to form endolysosomes, where degradation occurs. In some cases, both receptor and ligand are transferred to a different plasma membrane domain, causing the ligand to be released at a different surface from where it originated, a process called transcytosis. In some cells, endocytosed plasma membrane proteins and lipids can be stored in recycling endosomes, for as long as necessary until they are needed. TransporT froM The Trans golgI neTwork To The cell exTerIor: exocyTosIs


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Cell_Biology_Alberts. TransporT froM The Trans golgI neTwork To The cell exTerIor: exocyTosIs Having considered the cell’s endocytic and internal digestive systems and the various types of incoming membrane traffic that converge on lysosomes, we now return to the Golgi apparatus and examine the secretory pathways that lead outward to the cell exterior. Transport vesicles destined for the plasma membrane normally leave the TGN in a steady stream as irregularly shaped tubules. The membrane proteins and the lipids in these vesicles provide new components for the cell’s plasma membrane, while the soluble proteins inside the vesicles are secreted to the extracellular space. The fusion of the vesicles with the plasma membrane is called exocytosis. This is the route, for example, by which cells secrete most of the proteoglycans and glycoproteins of the extracellular matrix, as discussed in Chapter 19.


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Cell_Biology_Alberts. All cells require this constitutive secretory pathway, which operates continuously (Movie 13.6). Specialized secretory cells, however, have a second secretory pathway in which soluble proteins and other substances are initially stored in secretory vesicles for later release by exocytosis. This is the regulated secretory pathway, found mainly in cells specialized for secreting products rapidly on demand—such as hormones, neurotransmitters, or digestive enzymes (Figure 13–62). In this section, we consider the role of the Golgi apparatus in both of these pathways and compare the two mechanisms of secretion. Many proteins and lipids are carried automatically from the Trans golgi network (Tgn) to the cell surface


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Cell_Biology_Alberts. A cell capable of regulated secretion must separate at least three classes of proteins before they leave the TGN—those destined for lysosomes (via endosomes), those destined for secretory vesicles, and those destined for immediate delivery to the cell surface (Figure 13–63). We have already discussed how proteins destined for lysosomes are tagged with M6P for packaging into specific departing vesicles, and analogous signals are thought to direct secretory proteins into secretory vesicles. The nonselective constitutive secretory pathway transports most other proteins directly to the cell surface. Because entry into this pathway does not require a particular signal, it is also called the default pathway. Thus, in an unpolarized cell such as a white blood cell or a fibroblast, it seems that any protein in the lumen of the Golgi apparatus is automatically carried by the constitutive pathway to the cell surface unless it is specifically returned to the ER, retained as a resident protein in


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Cell_Biology_Alberts. protein in the lumen of the Golgi apparatus is automatically carried by the constitutive pathway to the cell surface unless it is specifically returned to the ER, retained as a resident protein in the Golgi apparatus itself, or selected for the pathways that lead to regulated secretion or to lysosomes. In polarized cells, where different products have to be delivered to different domains of the cell surface, we shall see that the options are more complex.


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Cell_Biology_Alberts. secretory vesicles bud from the Trans golgi network Cells that are specialized for secreting some of their products rapidly on demand concentrate and store these products in secretory vesicles (often called dense-core secretory granules because they have dense cores when viewed in the electron microscope). Secretory vesicles form from the TGN, and they release their contents to the cell exterior by exocytosis in response to specific signals. The secreted product can be either a small molecule (such as histamine or a neuropeptide) or a protein (such as a hormone or digestive enzyme).


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Cell_Biology_Alberts. Proteins destined for secretory vesicles (called secretory proteins) are packaged into appropriate vesicles in the TGN by a mechanism that involves the selective aggregation of the secretory proteins. Clumps of aggregated, electron-dense material can be detected by electron microscopy in the lumen of the TGN. The signals that direct secretory proteins into such aggregates are not well defined and may be quite diverse. When a gene encoding a secretory protein is artificially expressed in a secretory cell that normally does not make the protein, the foreign protein is appropriately packaged into secretory vesicles. This observation


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Cell_Biology_Alberts. Figure 13–62 The constitutive and regulated secretory pathways. The two pathways diverge in the Tgn. The constitutive secretory pathway operates in all cells. Many soluble proteins are continually secreted from the cell by this pathway, which also supplies the plasma membrane with newly synthesized membrane lipids and proteins. specialized secretory cells also have a regulated secretory pathway, by which selected proteins in the Tgn are diverted into secretory vesicles, where the proteins are concentrated and stored until an extracellular signal stimulates their secretion. The regulated secretion of small molecules, such as histamine and neurotransmitters, occurs by a similar pathway; these molecules are actively transported from the cytosol into preformed secretory vesicles. There they are often bound to specific macromolecules (proteoglycans, for histamine) so that they can be stored at high concentration without generating an excessively high osmotic pressure.


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Cell_Biology_Alberts. Figure 13–63 The three best-understood pathways of protein sorting in the trans Golgi network. (1) proteins with the mannose 6-phosphate (M6p) marker (see figure 13–45) are diverted to lysosomes (via endosomes) in clathrin-coated transport vesicles. (2) proteins with signals directing them to secretory vesicles are concentrated in such vesicles as part of a regulated secretory pathway that is present only in specialized secretory cells. (3) In unpolarized cells, a constitutive secretory pathway delivers proteins with no special features to the cell surface. In polarized cells, such as epithelial cells, however, secreted and plasma membrane proteins are selectively directed to either the apical or the basolateral plasma membrane domain, so a specific signal must mediate at least one of these two pathways, as we discuss later.


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Cell_Biology_Alberts. shows that, although the proteins that an individual cell expresses and packages in secretory vesicles differ, they contain common sorting signals, which function properly even when the proteins are expressed in cells that do not normally make them. It is unclear how the aggregates of secretory proteins are segregated into secretory vesicles. Secretory vesicles have unique proteins in their membrane, some of which might serve as receptors for aggregated protein in the TGN. The aggregates are much too big, however, for each molecule of the secreted protein to be bound by its own cargo receptor, as occurs for transport of the lysosomal enzymes. The uptake of the aggregates into secretory vesicles may therefore more closely resemble the uptake of particles by phagocytosis at the cell surface, where the plasma membrane zippers up around large structures.


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Cell_Biology_Alberts. Initially, most of the membrane of the secretory vesicles that leave the TGN is only loosely wrapped around the clusters of aggregated secretory proteins. Morphologically, these immature secretory vesicles resemble dilated trans Golgi cisternae that have pinched off from the Golgi stack. As the vesicles mature, they fuse with one another and their contents become concentrated (Figure 13–64A), probably as the result of both the continuous retrieval of membrane that is recycled to the TGN, and the progressive acidification of the vesicle lumen that results from the increasing concentration of V-type ATPases in the vesicle membrane that acidify all endocytic and exocytic organelles (see Figure 13–37). The degree of concentration of proteins during the formation and maturation of secretory vesicles is only a small part of the total 200–400-fold concentration of these proteins that occurs after they leave the ER. Secretory and membrane proteins become concentrated as they move from the ER


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Cell_Biology_Alberts. is only a small part of the total 200–400-fold concentration of these proteins that occurs after they leave the ER. Secretory and membrane proteins become concentrated as they move from the ER through the Golgi apparatus because of an extensive retrograde retrieval process mediated by COPI-coated transport vesicles that carry soluble ER resident proteins back to the ER, while excluding the secretory and membrane proteins (see Figure 13–25).


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Cell_Biology_Alberts. Membrane recycling is important for returning Golgi components to the Golgi apparatus, as well as for concentrating the contents of secretory vesicles. The vesicles that mediate this retrieval originate as clathrin-coated buds on the surface of immature secretory vesicles, often being seen even on budding secretory vesicles that have not yet pinched off from the Golgi stack (Figure 13–64B). Because the final mature secretory vesicles are so densely filled with contents, the secretory cell can disgorge large amounts of material promptly by exocytosis when triggered to do so (Figure 13–65). precursors of secretory proteins are proteolytically processed during the formation of secretory vesicles Concentration is not the only process to which secretory proteins are subjected as the secretory vesicles mature. Many protein hormones and small neuropeptides,


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Cell_Biology_Alberts. Figure 13–64 The formation of secretory vesicles. (a) secretory proteins become segregated and highly concentrated in secretory vesicles by two mechanisms. first, they aggregate in the ionic environment of the Tgn; often the aggregates become more condensed as secretory vesicles mature and their lumen becomes more acidic. second, clathrincoated vesicles retrieve excess membrane and lumenal content present in immature secretory vesicles as the secretory vesicles mature. (b) This electron micrograph shows secretory vesicles forming from the Tgn in an insulin-secreting β cell of the pancreas. anti-clathrin antibodies conjugated to gold spheres (black dots) have been used to locate clathrin molecules. The immature secretory vesicles, which contain insulin precursor protein (proinsulin), contain clathrin patches, which are no longer seen on the mature secretory vesicle. (b, courtesy of lelio orci.) as well as many secreted hydrolytic enzymes, are synthesized as inactive precursors.


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Cell_Biology_Alberts. contain clathrin patches, which are no longer seen on the mature secretory vesicle. (b, courtesy of lelio orci.) as well as many secreted hydrolytic enzymes, are synthesized as inactive precursors. Proteolysis is necessary to liberate the active molecules from these precursor proteins. The cleavages occur in the secretory vesicles and sometimes in the extracellular fluid after secretion. Additionally, many of the precursor proteins have an N-terminal pro-peptide that is cleaved off to yield the mature protein. These proteins are synthesized as pre-pro-proteins, the pre-peptide consisting of the ER signal peptide that is cleaved off earlier in the rough ER (see Figure 12–36). In other cases, peptide signaling molecules are made as polyproteins that contain multiple copies of the same amino acid sequence. In still more complex cases, a variety of peptide signaling molecules are synthesized as parts of a single polyprotein that acts as a precursor for multiple end products, which are


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Cell_Biology_Alberts. acid sequence. In still more complex cases, a variety of peptide signaling molecules are synthesized as parts of a single polyprotein that acts as a precursor for multiple end products, which are individually cleaved from the initial polypeptide chain. The same polyprotein may be processed in various ways to produce different peptides in different cell types (Figure 13–66).


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Cell_Biology_Alberts. Why is proteolytic processing so common in the secretory pathway? Some of the peptides produced in this way, such as the enkephalins (five-amino-acid neuropeptides with morphine-like activity), are undoubtedly too short in their mature forms to be co-translationally transported into the ER lumen or to include the necessary signal for packaging into secretory vesicles. In addition, for secreted hydrolytic enzymes—or any other protein whose activity could be harmful inside the cell that makes it—delaying activation of the protein until it reaches a secretory vesicle, or until after it has been secreted, has a clear advantage: the delay prevents the protein from acting prematurely inside the cell in which it is synthesized. secretory vesicles wait near the plasma Membrane until signaled to release Their contents


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Cell_Biology_Alberts. secretory vesicles wait near the plasma Membrane until signaled to release Their contents Once loaded, a secretory vesicle has to reach the site of secretion, which in some cells is far away from the TGN. Nerve cells are the most extreme example. Secretory proteins, such as peptide neurotransmitters (neuropeptides), which will be released from nerve terminals at the end of the axon, are made and packaged into secretory vesicles in the cell body. They then travel along the axon to the nerve terminals, which can be a meter or more away. As discussed in Chapter 16, motor proteins propel the vesicles along axonal microtubules, whose uniform orientation guides the vesicles in the proper direction. Microtubules also guide transport vesicles to the cell surface for constitutive exocytosis.


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Cell_Biology_Alberts. Whereas transport vesicles containing materials for constitutive release fuse with the plasma membrane once they arrive there, secretory vesicles in the regulated pathway wait at the membrane until the cell receives a signal to secrete, and they then fuse. The signal can be an electrical nerve impluse (an action potential) or an extracellular signal molecule, such as a hormone: in either case, it leads to a transient increase in the concentration of free Ca2+ in the cytosol. for rapid exocytosis, synaptic vesicles are primed at the presynaptic plasma Membrane Nerve cells (and some endocrine cells) contain two types of secretory vesicles. As for all secretory cells, these cells package proteins and neuropeptides in dense-cored secretory vesicles in the standard way for release by the regulated secretory pathway. In addition, however, they use another specialized class of tiny (≈50 nm 0.2 µm


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Cell_Biology_Alberts. Figure 13–65 Exocytosis of secretory vesicles. The process is illustrated schematically (top) and in an electron micrograph that shows the release of insulin from a secretory vesicle of a pancreatic β cell. (courtesy of lelio orci, from l. orci, J.-d. vassalli and a. perrelet, sci. am. 259:85–94, 1988.)


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Cell_Biology_Alberts. Figure 13–66 Alternative processing pathways for the prohormone polyprotein proopiomelanocortin. The initial cleavages are made by proteases that cut next to pairs of positively charged amino acids (lys-arg, lys-lys, arg-lys, or arg-arg pairs). Trimming reactions then produce the final secreted products. different cell types produce different concentrations of individual processing enzymes, so that the same prohormone precursor is cleaved to produce different peptide hormones. In the anterior lobe of the pituitary gland, for example, only corticotropin (acTh) and β-lipotropin are produced from proopiomelanocortin, whereas in the intermediate lobe of the pituitary gland mainly α-melanocyte stimulating hormone (α-Msh), γ-lipotropin, β-Msh, and β-endorphin are produced— α-Msh from acTh and the other three from β-lipotropin, as shown. presynaptic (B) 1. DOCKING presynaptic 2. PRIMING I partially plasma membrane plasma membrane


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Cell_Biology_Alberts. presynaptic (B) 1. DOCKING presynaptic 2. PRIMING I partially plasma membrane plasma membrane SNARE bundle 3. PRIMING II4. FUSION PORE OPENING5. FUSION COMPLETE complexin released complexin-Ca2+


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Cell_Biology_Alberts. Figure 13–67 Exocytosis of synaptic vesicles. for orientation at a synapse, see figure 11–36. (a) The trans-snare complex responsible for docking synaptic vesicles at the plasma membrane of nerve terminals consists of three proteins. The v-snare synaptobrevin and the t-snare syntaxin are both transmembrane proteins, and each contributes one α helix to the complex. by contrast to other snares discussed earlier, the t-snare snaP25 is a peripheral membrane protein that contributes two α helices to the four-helix bundle; the two helices are connected by a loop (dashed line) that lies parallel to the membrane and has fatty acyl chains (not shown) attached to anchor it there. The four α helices are shown as rods for simplicity. (b) at the synapse, the basic snare machinery is modulated by the ca2+ sensor synaptotagmin and an additional protein called complexin. synaptic vesicles first dock at the membrane (step 1), and the snare bundle partially assembles (step 2), resulting in a “primed


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Cell_Biology_Alberts. ca2+ sensor synaptotagmin and an additional protein called complexin. synaptic vesicles first dock at the membrane (step 1), and the snare bundle partially assembles (step 2), resulting in a “primed vesicle” that is already drawn close to the membrane. The snare bundle assembles further but the additional binding of complexin prevents fusion (step 3). upon arrival of an action potential, ca2+ enters the cell and binds to synaptotagmin, which releases the block and opens a fusion pore (step 4). further rearrangements complete the fusion reaction (step 5) and release the fusion machinery, which now can be reused. This elaborate arrangement allows the fusion machinery to respond on the millisecond time scale essential for rapid and repetitive synaptic signaling. (a, adapted from r.b. sutton et al., nature 395:347–353, 1998. with permission from Macmillan publishers ltd.; b, adapted from Z.p. pang and T.c. südhof, Curr. Opin. Cell Biol. 22:496–505, 2010. with permission from elsevier.)


Cell_Biology_Alberts

Cell_Biology_Alberts. et al., nature 395:347–353, 1998. with permission from Macmillan publishers ltd.; b, adapted from Z.p. pang and T.c. südhof, Curr. Opin. Cell Biol. 22:496–505, 2010. with permission from elsevier.) diameter) secretory vesicles called synaptic vesicles. These vesicles store small neurotransmitter molecules, such as acetylcholine, glutamate, glycine, and γ-aminobutyric acid (GABA), which mediate rapid signaling from nerve cell to its target cell at chemical synapses. When an action potential arrives at a nerve terminal, it causes an influx of Ca2+ through voltage-gated Ca2+ channels, which triggers the synaptic vesicles to fuse with the plasma membrane and release their contents to the extracellular space (see Figure 11–36). Some neurons fire more than 1000 times per second, releasing neurotransmitters each time.


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Cell_Biology_Alberts. The speed of transmitter release (taking only milliseconds) indicates that the proteins mediating the fusion reaction do not undergo complex, multistep rearrangements. Rather, after vesicles have been docked at the presynaptic plasma membrane, they undergo a priming step, which prepares them for rapid fusion. In the primed state, the SNAREs are partly paired, their helices are not fully wound into the final four-helix bundle required for fusion (Figure 13–67). Proteins called complexins freeze the SNARE complexes in this metastable state. The brake imposed by the complexins is released by another synaptic vesicle protein, synaptotagmin, which contains Ca2+-binding domains. A rise in cytosolic Ca2+ triggers binding of synaptotagmin to phospholipids and to the SNAREs, displacing the complexins. As the SNARE bundle zippers up completely, a fusion pore opens and the neurotransmitters are released. At a typical synapse, only a small number of the docked vesicles are primed and ready for


Cell_Biology_Alberts

Cell_Biology_Alberts. As the SNARE bundle zippers up completely, a fusion pore opens and the neurotransmitters are released. At a typical synapse, only a small number of the docked vesicles are primed and ready for exocytosis. The use of only a small number of vesicles at a time allows each synapse to fire over and over again in quick succession. With each firing, new synaptic vesicles dock and become primed to replace those that have fused and released their contents.


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Cell_Biology_Alberts. For the nerve terminal to respond rapidly and repeatedly, synaptic vesicles need to be replenished very quickly after they discharge. Thus, most synaptic vesicles are generated not from the Golgi membrane in the nerve cell body but by local recycling from the presynaptic plasma membrane in the nerve terminals (Figure 13–68). Similarly, newly made membrane components of the synaptic vesicles are initially delivered to the plasma membrane by the constitutive secretory pathway and then retrieved by endocytosis. But instead of fusing with endosomes, most of the endocytic vesicles immediately fill with neurotransmitter to become synaptic vesicles.


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Cell_Biology_Alberts. The membrane components of a synaptic vesicle include transporters specialized for the uptake of neurotransmitter from the cytosol, where the small-molecule neurotransmitters that mediate fast synaptic signaling are synthesized. Once filled with neurotransmitter, the synaptic vesicles can be used again (see Figure 13–68). Because synaptic vesicles are abundant and relatively uniform in size, they can be purified in large numbers and, consequently, are the best-characterized organelle of the cell, in that all of their membrane components have been identified by quantitative proteomic analyses (Figure 13–69). Extending this analysis to a complete presynaptic terminal, allows us to model the crowded environment in which these reactions occur. secretory vesicle Membrane components are Quickly removed from the plasma Membrane


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Cell_Biology_Alberts. secretory vesicle Membrane components are Quickly removed from the plasma Membrane When a secretory vesicle fuses with the plasma membrane, its contents are discharged from the cell by exocytosis, and its membrane becomes part of the plasma membrane. Although this should greatly increase the surface area of the plasma membrane, it does so only transiently, because membrane components are removed from the surface by endocytosis almost as fast as they are added by exocytosis, a process reminiscent of the endocytic–exocytic cycle discussed earlier. After their removal from the plasma membrane, the proteins of the secretory


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Cell_Biology_Alberts. Figure 13–68 The formation of synaptic vesicles in a nerve cell. These tiny uniform vesicles are found only in nerve cells and in some endocrine cells, where they store and secrete small-molecule neurotransmitters. The import of neurotransmitter directly into the small endocytic vesicles that form from the plasma membrane is mediated by membrane transporters that function as antiports and are driven by an h+ gradient maintained by v-aTpase h+ pumps in the vesicle membrane (discussed in chapter 11).


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Cell_Biology_Alberts. Figure 13–69 Scale models of a brain presynaptic terminal and a synaptic vesicle. The illustrations show sections through a pre-synaptic terminal (a; enlarged in b) and a synaptic vesicle (c) in which proteins and lipids are drawn to scale based on their known stoichiometry and either known or approximated structures. The relative localization of protein molecules in different regions of the presynaptic terminal was inferred from super-resolution imaging and electron microscopy. The model in (a) contains 300,000 proteins of 60 different kinds that vary in abundance from 150 copies to 20,000 copies. In the model in (c), only 70% of the membrane proteins present in the membrane are shown; a complete model would therefore show a membrane that is even more crowded than this picture suggests (Movie 13.7). each synaptic vesicle membrane contains 7000 phospholipid molecules and 5700 cholesterol molecules. each also contains close to 50 different integral membrane protein molecules, which


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Cell_Biology_Alberts. (Movie 13.7). each synaptic vesicle membrane contains 7000 phospholipid molecules and 5700 cholesterol molecules. each also contains close to 50 different integral membrane protein molecules, which vary widely in their relative abundance and together contribute about 600 transmembrane α helices. The transmembrane v-snare synaptobrevin is the most abundant protein in the vesicle (~70 copies per vesicle). by contrast, the v-aTpase, which uses aTp hydrolysis to pump h+ into the vesicle lumen, is present in 1–2 copies per vesicle. The h+ gradient provides the energy for neurotransmitter import by an h+/neurotransmitter antiport, which loads each vesicle with 1800 neurotransmitter molecules, such as glutamate, one of which is shown to scale. (a and b, from b.g. wilhelm et al., science 344:1023–1028, 2014. with permission from aaas; c, adapted from s. Takamori et al., Cell 127:831–846, 2006. with permission from elsevier.) vesicle membrane are either recycled or shuttled to lysosomes for


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Cell_Biology_Alberts. 2014. with permission from aaas; c, adapted from s. Takamori et al., Cell 127:831–846, 2006. with permission from elsevier.) vesicle membrane are either recycled or shuttled to lysosomes for degradation. The amount of secretory vesicle membrane that is temporarily added to the plasma membrane can be enormous: in a pancreatic acinar cell discharging digestive enzymes for delivery to the gut lumen, about 900 μm2 of vesicle membrane is inserted into the apical plasma membrane (whose area is only 30 μm2) when the cell is stimulated to secrete.