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Cell_Biology_Alberts. Nuclear localization signals can be located almost anywhere in the amino acid sequence and are thought to form loops or patches on the protein surface. Many function even when linked as short peptides to lysine side chains on the surface of a cytosolic protein, suggesting that the precise location of the signal within the amino acid sequence of a nuclear protein is not important. Moreover, as long as one of the protein subunits of a multicomponent complex displays a nuclear localization signal, the entire complex will be imported into the nucleus. Figure 12–7 The nuclear envelope. The double-membrane envelope is penetrated by pores in which nuclear pore complexes (not shown) are positioned. The outer nuclear membrane is continuous with the endoplasmic reticulum (er). The ribosomes that are normally bound to the cytosolic surface of the er membrane and outer nuclear membrane are not shown. The nuclear lamina is a fibrous protein meshwork underlying the inner membrane.


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Cell_Biology_Alberts. One can visualize the transport of nuclear proteins through NPCs by coating gold particles with a nuclear localization signal, injecting the particles into the cytosol, and then following their fate by electron microscopy (Figure 12–10). The particles bind to the tentaclelike fibrils that extend from the scaffold nucleoporins at the rim of the NPC into the cytosol, and then proceed through the center of the NPC. Presumably, the unstructured regions of the nucleoporins that form a diffusion barrier for large molecules (mentioned earlier) are pushed away to allow the coated gold particles to squeeze through. Macromolecular transport across NPCs differs fundamentally from the transport of proteins across the membranes of other organelles, in that it occurs (B) 0.1 µm inner nuclear membrane nuclear envelope outer nuclear membrane 50 nm scaffold nucleoporins membrane ring proteins channel nucleoporinsnuclear basket disordered region of channel nucleoporins cytosolic fbrils 0.1 µm 0.1 µm


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Cell_Biology_Alberts. Figure 12–8 The arrangement of NPCs in the nuclear envelope. (a) in a vertebrate npC, nucleoporins are arranged with striking eightfold rotational symmetry. in addition, immunoelectron microscopic studies show that the proteins that make up the central portion of the npC are oriented symmetrically across the nuclear envelope, so that the nuclear and cytosolic sides look identical. The eightfold rotational and twofold transverse symmetry explains how such a huge structure can be formed from only about 30 different proteins: many of the nucleoporins are present in 8, 16, or 32 copies. Based on their approximate localization in the central portion of the npC, nucleoporins can be classified into (1) transmembrane ring proteins that span the nuclear envelope and anchor the npC to the envelope; (2) scaffold nucleoporins that form layered ring structures. some scaffold nucleoporins are membrane-bending proteins that stabilize the sharp membrane curvature where the nuclear envelope is


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Cell_Biology_Alberts. (2) scaffold nucleoporins that form layered ring structures. some scaffold nucleoporins are membrane-bending proteins that stabilize the sharp membrane curvature where the nuclear envelope is penetrated; and (3) channel nucleoporins that line a central pore. in addition to folded domains that anchor the proteins in specific places, many channel nucleoporins contain extensive unstructured regions, where the polypeptide chains are intrinsically disordered. The central pore is filled with a tangled mesh of these disordered domains that blocks the passive diffusion of large macromolecules. The disordered regions contain a large number of phenylalanine-glycine (fG) repeats. fibrils protrude from both the cytosolic and the nuclear sides of the npC. By contrast to the twofold transverse symmetry of the npC core, the fibrils facing the cytosol and nucleus are different: on the nuclear side, the fibrils converge at their distal end to form a basketlike structure. The precise arrangement of


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Cell_Biology_Alberts. of the npC core, the fibrils facing the cytosol and nucleus are different: on the nuclear side, the fibrils converge at their distal end to form a basketlike structure. The precise arrangement of individual nucleoporins in the assembled npC is still a matter of intense debate, because atomic resolution analyses have been hindered by the sheer size and flexible nature of the npC, and by difficulties in purifying sufficient amounts of homogeneous material. a combination of electron microscopy, computational analyses, and crystal structures of nucleoporin subcomplexes has been used to develop the current models of the npC architecture. (B) a scanning electron micrograph of the nuclear side of the nuclear envelope of an oocyte (see also figure 9–52). (C) an electron micrograph showing a side view of two npCs (brackets); note that the inner and outer nuclear membranes are continuous at the edges of the pore. (D) an electron micrograph showing face-on views of negatively stained npCs. The


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Cell_Biology_Alberts. view of two npCs (brackets); note that the inner and outer nuclear membranes are continuous at the edges of the pore. (D) an electron micrograph showing face-on views of negatively stained npCs. The membrane has been removed by detergent extraction. note that some of the npCs contain material in their center, which is thought to be trapped macromolecules in transit through these npCs. (a, adapted from a. hoelz, e.W. Debler and G. Blobel, Annu. Rev. Biochem. 80:613– 643, 2011. With permission from annual reviews; B, from m.W. Goldberg and T.D. allen, J. Cell Biol. 119:1429–1440, 1992. With permission from The rockefeller University press; C, courtesy of Werner franke and Ulrich scheer; D, courtesy of ron milligan.) through a large, expandable, aqueous pore, rather than through a protein transporter spanning one or more lipid bilayers. For this reason, fully folded nuclear proteins can be transported into the nucleus through an NPC, and newly formed ribosomal subunits are transported


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Cell_Biology_Alberts. transporter spanning one or more lipid bilayers. For this reason, fully folded nuclear proteins can be transported into the nucleus through an NPC, and newly formed ribosomal subunits are transported out of the nucleus as an assembled particle. By contrast, proteins have to be extensively unfolded to be transported into most other organelles, as we discuss later.


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Cell_Biology_Alberts. nuclear import receptors Bind to Both nuclear localization signals and npC proteins To initiate nuclear import, most nuclear localization signals must be recognized by nuclear import receptors, sometimes called importins, most of which are encoded by a family of related genes. Each family member encodes a receptor protein that can bind and transport the subset of cargo proteins containing the appropriate nuclear localization signal (Figure 12–11A). Nuclear import receptors do not always bind to nuclear proteins directly. Additional adaptor proteins can form a bridge between the import receptors and the nuclear localization signals on the proteins to be transported (Figure 12–11B). Some adaptor proteins are structurally related to nuclear import receptors, suggesting a common evolutionary origin. By using a variety of import receptors and adaptors, cells are able to recognize the broad repertoire of nuclear localization signals that are displayed on nuclear proteins.


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Cell_Biology_Alberts. The import receptors are soluble cytosolic proteins that bind both to the nuclear localization signal on the cargo protein and to the phenylalanine-glycine (FG) repeats in the unstructured domains of the channel nucleoporins that line the central pore. FG-repeats are also found in the cytoplasmic and nuclear fibrils. FG-repeats in the unstructured tangle of the pore are thought to do double duty. They interact weakly, which gives the protein tangle gel-like properties that impose a permeability barrier to large macromolecules, and they serve as docking sites for nuclear import receptors. FG-repeats line the path through the NPCs taken by the import receptors and their bound cargo proteins. According to one model of nuclear transport, the receptor–cargo complexes move along the transport path by repeatedly binding, dissociating, and then re-binding to adjacent FG-repeat sequences. In this way, the complexes may hop from one nucleoporin to another to traverse the tangled interior of the


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Cell_Biology_Alberts. by repeatedly binding, dissociating, and then re-binding to adjacent FG-repeat sequences. In this way, the complexes may hop from one nucleoporin to another to traverse the tangled interior of the NPC in a random walk. As import receptors bind to FG-repeats during this journey, they would disrupt interaction between the repeats and locally dissolve the gel phase of the protein tangle that fills the pore, allowing the passage of the receptor–cargo complex. Once inside the nucleus, the import receptors dissociate from their cargo and return to the cytosol. As we will see, this dissociation only occurs on the nuclear side of the NPC and thereby confers directionality to the import process.


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Cell_Biology_Alberts. nuclear export Works like nuclear import, But in reverse The nuclear export of large molecules, such as new ribosomal subunits and RNA molecules, occurs through NPCs and also depends on a selective transport Figure 12–9 The function of a nuclear localization signal. immunofluorescence micrographs showing the cell location of sV40 virus T-antigen containing or lacking a short sequence that serves as a nuclear localization signal. (a) The normal T-antigen protein contains the lysine-rich sequence indicated and is imported to its site of action in the nucleus, as indicated by immunofluorescence staining with antibodies against the T-antigen. T-antigen with an altered nuclear localization signal (a threonine replacing a lysine) remains in the cytosol. (from D. Kalderon, B. roberts, W. richardson and a. smith, Cell 39:499–509, 1984. With permission from elsevier.)


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Cell_Biology_Alberts. Figure 12–10 Visualizing active import through NPCs. This series of electron micrographs shows colloidal gold spheres (arrowheads) coated with peptides containing nuclear localization signals entering the nucleus through npCs. The gold particles were injected into the cytosol of living cells, which then were fixed and prepared for electron microscopy at various times after injection. (a) Gold particles are first seen in proximity to the cytosolic fibrils of the npCs. (B, C) They are then seen at the center of the npCs, exclusively on the cytosolic face. (D) They then appear on the nuclear face. These gold particles have much larger diameters than the diffusion channels in the npC and are imported by active transport. (from n. panté and U. aebi, Science 273:1729–1732, 1996. With permission from aaas.) system. The transport system relies on nuclear export signals on the macromolecules to be exported, as well as on complementary nuclear export receptors, or exportins. These receptors


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Cell_Biology_Alberts. from aaas.) system. The transport system relies on nuclear export signals on the macromolecules to be exported, as well as on complementary nuclear export receptors, or exportins. These receptors bind to both the export signal and NPC proteins to guide their cargo through the NPC to the cytosol.


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Cell_Biology_Alberts. Many nuclear export receptors are structurally related to nuclear import receptors, and they are encoded by the same gene family of nuclear transport receptors, or karyopherins. In yeast, there are 14 genes encoding karyopherins; in animal cells, the number is significantly larger. It is often not possible to tell from their amino acid sequence alone whether a particular family member works as a nuclear import or nuclear export receptor. As might be expected, therefore, the import and export transport systems work in similar ways but in opposite directions: the import receptors bind their cargo molecules in the cytosol, release them in the nucleus, and are then exported to the cytosol for reuse, while the export receptors function in the opposite fashion. The ran GTpase imposes Directionality on Transport Through npCs


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Cell_Biology_Alberts. The ran GTpase imposes Directionality on Transport Through npCs The import of nuclear proteins through NPCs concentrates specific proteins in the nucleus and thereby increases order in the cell. The cell fuels this ordering process by harnessing energy stored in concentration gradients of the GTP-bound form of the monomeric GTPase Ran, which is required for both nuclear import and export.


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Cell_Biology_Alberts. Like other GTPases, Ran is a molecular switch that can exist in two conformational states, depending on whether GDP or GTP is bound (discussed in Chapter 3). Two Ran-specific regulatory proteins trigger the conversion between the two states: a cytosolic GTPase-activating protein (GAP) triggers GTP hydrolysis and thus converts Ran-GTP to Ran-GDP, and a nuclear guanine exchange factor (GEF) promotes the exchange of GDP for GTP and thus converts Ran-GDP to Ran-GTP. Because Ran-GAP is located in the cytosol and Ran-GEF is located in the nucleus where it is anchored to chromatin, the cytosol contains mainly Ran-GDP, and the nucleus contains mainly Ran-GTP (Figure 12–12).


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Cell_Biology_Alberts. Figure 12–11 Nuclear import receptors (importins). (a) Different nuclear import receptors bind different nuclear localization signals and thereby different cargo proteins. (B) Cargo protein 4 requires an adaptor protein to bind to its nuclear import receptor. The adaptors are structurally related to nuclear import receptors and recognize nuclear localization signals on cargo proteins. They also contain a nuclear localization signal that binds them to an import receptor, but this signal only becomes exposed when they are loaded with a cargo protein. Figure 12–12 The compartmentalization of Ran-GDP and Ran-GTP. localization of ran-GDp in the cytosol and ran-GTp in the nucleus results from the localization of two ran regulatory proteins: ran GTpaseactivating protein (ran-Gap) is located in the cytosol, and ran guanine nucleotide exchange factor (ran-Gef) binds to chromatin and is therefore located in the nucleus.


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Cell_Biology_Alberts. ran-GDp is imported into the nucleus by its own import receptor, which is specific for the GDp-bound conformation of ran. The ran-GDp receptor is structurally unrelated to the main family of nuclear transport receptors. however, it also binds to fG-repeats in npC channel nucleoporins. 654 Chapter 12: intracellular Compartments and protein sorting


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Cell_Biology_Alberts. 654 Chapter 12: intracellular Compartments and protein sorting This gradient of the two conformational forms of Ran drives nuclear transport in the appropriate direction. Docking of nuclear import receptors to FG-repeats on the cytosolic side of the NPC, for example, occurs whether or not these receptors are loaded with appropriate cargo. Import receptors, facilitated by FG-repeat binding, then enter the channel. If they reach the nuclear side of the pore complex, Ran-GTP binds to them, and, if the receptors arrive loaded with cargo molecules, the Ran-GTP binding causes the receptors to release their cargo (Figure 12–13A). Because the Ran-GDP in the cytosol does not bind to import (or export) receptors, unloading occurs only on the nuclear side of the NPC. In this way, the nuclear localization of Ran-GTP creates the directionality of the import process.


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Cell_Biology_Alberts. Having discharged its cargo in the nucleus, the empty import receptor with Ran-GTP bound is transported back through the pore complex to the cytosol. There, Ran-GAP triggers Ran-GTP to hydrolyze its bound GTP, thereby converting it to Ran-GDP, which dissociates from the receptor. The receptor is then ready for another cycle of nuclear import. Nuclear export occurs by a similar mechanism, except that Ran-GTP in the nucleus promotes cargo binding to the export receptor, rather than promoting cargo dissociation. Once the export receptor moves through the pore to the cytosol, it encounters Ran-GAP, which induces the receptor to hydrolyze its GTP to GDP. As a result, the export receptor releases both its cargo and Ran-GDP in the cytosol. Free export receptors are then returned to the nucleus to complete the cycle (Figure 12–13B). Transport Through npCs Can Be regulated by Controlling access to the Transport machinery


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Cell_Biology_Alberts. Transport Through npCs Can Be regulated by Controlling access to the Transport machinery Some proteins contain both nuclear localization signals and nuclear export signals. These proteins continually shuttle back and forth between the nucleus and the cytosol. The relative rates of their import and export determine the steady-state localization of such shuttling proteins: if the rate of import exceeds the rate of export, a protein will be located mainly in the nucleus; conversely, if the rate of export exceeds the rate of import, a protein will be located mainly in the cytosol. Thus, changing the rate of import, export, or both, can change the location of a protein. Figure 12–13 How GTP hydrolysis by Ran in the cytosol provides directionality to nuclear transport.


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Cell_Biology_Alberts. Figure 12–13 How GTP hydrolysis by Ran in the cytosol provides directionality to nuclear transport. movement through the npC of loaded nuclear transport receptors occurs along the fG-repeats displayed by certain npC proteins. The differential localization of ran-GTp in the nucleus and ran-GDp in the cytosol provides directionality (red arrows) to both nuclear import (a) and nuclear export (B). ran-Gap stimulates the hydrolysis of GTp to produce ran-GDp on the cytosolic side of the npC (see figure 12–12).


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Cell_Biology_Alberts. Some shuttling proteins move continuously into and out of the nucleus. In other cases, however, the transport is stringently controlled. As discussed in Chapter 7, cells control the activity of some transcription regulators by keeping them out of the nucleus until they are needed there (Figure 12–14). In many cases, cells control transport by regulating nuclear localization and export signals—turning them on or off, often by phosphorylation of amino acids close to the signal sequences (Figure 12–15).


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Cell_Biology_Alberts. Other transcription regulators are bound to inhibitory cytosolic proteins that either anchor them in the cytosol (through interactions with the cytoskeleton or specific organelles) or mask their nuclear localization signals so that they cannot interact with nuclear import receptors. An appropriate stimulus releases the gene regulatory protein from its cytosolic anchor or mask, and it is then transported into the nucleus. One important example is the latent gene regulatory protein that controls the expression of proteins involved in cholesterol metabolism. The protein is made and stored in an inactive form as a transmembrane protein in the ER. When a cell is deprived of cholesterol, the protein is transported from the ER to the Golgi apparatus where it encounters specific proteases that cleave off the cytosolic domain, releasing it into the cytosol. This domain is then imported into the nucleus, where it activates the transcription of genes required for both cholesterol uptake and


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Cell_Biology_Alberts. cleave off the cytosolic domain, releasing it into the cytosol. This domain is then imported into the nucleus, where it activates the transcription of genes required for both cholesterol uptake and synthesis (Figure 12–16).


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Cell_Biology_Alberts. As we discuss in detail in Chapter 6, cells control the export of RNAs from the nucleus in a similar way. snRNAs, miRNAs, and tRNAs bind to the same family of nuclear export receptors just discussed, and they use the same Ran-GTP gradient to fuel the transport process. By contrast, the export of mRNAs out of the nucleus uses a different mechanism. mRNAs are exported as large assemblies, which can be as large as 100 million daltons (see Figure 6–37) and can contain hundreds of proteins of a few dozen different types. These mRNA ribonucleoprotein complexes (mRNPs) first dock at the nuclear side of the NPC, where they are extensively remodeled. Although Ran-GTP is indirectly involved in the export (because it imports the proteins that bind to the mRNA molecules), the translocation across the NPC is thought to be driven by ATP hydrolysis. How export directionality is assured is unclear. It is likely that the many accessory proteins tethered to the NPC’s nuclear and cytoplasmic fibrils


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Cell_Biology_Alberts. the NPC is thought to be driven by ATP hydrolysis. How export directionality is assured is unclear. It is likely that the many accessory proteins tethered to the NPC’s nuclear and cytoplasmic fibrils have important roles in remodeling the mRNPs as they pass through the pores, in particular stripping away nuclear proteins as the mRNPs exit on the cytosolic side of the NPC, thereby ensuring that transport is unidirectional. Upon entry into the cytosol, these nuclear mRNP


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Cell_Biology_Alberts. Figure 12–14 The control of nuclear transport in the early Drosophila embryo. The embryo at this stage is a syncytium, shown here in cross section, with many nuclei in a common cytoplasm, arranged around the periphery, just beneath the plasma membrane. The transcription regulatory protein Dorsal is produced uniformly throughout the peripheral cytoplasm, but it can act only when inside the nuclei. The Dorsal protein has been stained with an enzyme-coupled antibody that yields a brown product, revealing that Dorsal is excluded from the nuclei at the dorsal side (top) of the embryo but is concentrated in the nuclei toward the ventral side (bottom) of the embryo. The regulated traffic of Dorsal into the nuclei controls the differential development between the back and belly of the animal. (Courtesy of siegfried roth.)


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Cell_Biology_Alberts. Figure 12–15 The control of nuclear import during T cell activation. The nuclear factor of activated T cells (nf-aT) is a transcription regulatory protein that, in the resting T cell, is found in the cytosol in a phosphorylated state. When T cells are activated by foreign antigen (discussed in Chapter 24), the intracellular Ca2+ concentration increases. in high Ca2+, the protein phosphatase calcineurin binds to nf-aT and dephosphorylates it. The dephosphorylation exposes nuclear import signals and blocks a nuclear export signal. The complex of nf-aT and calcineurin is therefore imported into the nucleus, where nf-aT activates the transcription of numerous genes required for T cell activation.


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Cell_Biology_Alberts. The response shuts off when Ca2+ levels decrease, releasing nf-aT from calcineurin. rephosphorylation of nf-aT inactivates the nuclear import signals and re-exposes the nuclear export signal, causing nf-aT to relocate to the cytosol. some of the most potent immunosuppressive drugs, including cyclosporin a and fK506, inhibit the ability of calcineurin to dephosphorylate nf-aT and thereby block the nuclear accumulation of nf-aT and T cell activation (Movie 12.1). During mitosis the nuclear envelope Disassembles


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Cell_Biology_Alberts. During mitosis the nuclear envelope Disassembles The nuclear lamina, located on the nuclear side of the inner nuclear membrane, is a meshwork of interconnected protein subunits called nuclear lamins. The lamins are a special class of intermediate filament proteins (discussed in Chapter 16) that polymerize into a two-dimensional lattice (Figure 12–17). The nuclear lamina gives shape and stability to the nuclear envelope, to which it is anchored by attachment to both the NPCs and transmembrane proteins of the inner nuclear membrane. The lamina also interacts directly with chromatin, which itself interacts with transmembrane proteins of the inner nuclear membrane. Together with the lamina, these inner membrane proteins provide structural links between the DNA and the nuclear envelope.


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Cell_Biology_Alberts. When a nucleus is dismantled during mitosis, the NPCs and nuclear lamina disassemble and the nuclear envelope fragments. The dismantling process is at least partly a consequence of direct phosphorylation of nucleoporins and lamins by the cyclin-dependent protein kinase (Cdk) that is activated at the onset of mitosis (discussed in Chapter 17). During this process, some NPC proteins become bound to nuclear import receptors, which play an important part in the reassembly of NPCs at the end of mitosis. Nuclear envelope membrane proteins—no longer tethered to the pore complexes, lamina, or chromatin—disperse throughout the ER membrane. The dynein motor protein, which moves along microtubules (discussed in Chapter 16), actively participates in tearing the nuclear envelope off the chromatin. Together, these processes break down the barriers that normally separate the nucleus and cytosol, and the nuclear proteins that are not bound to membranes or chromosomes intermix completely with the


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Cell_Biology_Alberts. Together, these processes break down the barriers that normally separate the nucleus and cytosol, and the nuclear proteins that are not bound to membranes or chromosomes intermix completely with the proteins of the cytosol (Figure 12–18).


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Cell_Biology_Alberts. Later in mitosis, the nuclear envelope reassembles on the surface of the daughter chromosomes. In addition to its crucial role in nuclear transport, the Ran GTPase also acts as a positional marker for chromatin during cell division, when the nuclear and cytosolic components intermix. Because Ran-GEF remains bound to chromatin when the nuclear envelope breaks down, Ran molecules close to chromatin are mainly in their GTP-bound conformation. By contrast, Ran molecules further away have a high likelihood of encountering Ran-GAP, which is distributed throughout the cytosol; these Ran molecules are mainly in their GDP-bound conformation. As a result, the chromosomes in mitotic cells are surrounded by a cloud of Ran-GTP. Ran-GTP releases the NPC proteins in proximity to the chromosomes from nuclear import receptors. The free NPC proteins attach


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Cell_Biology_Alberts. Figure 12–16 Feedback regulation of cholesterol biosynthesis. sreBp (sterol response element binding protein), a latent transcription regulator that controls expression of cholesterol biosynthetic enzymes, is initially synthesized as an er membrane protein. it is anchored in the er if there is sufficient cholesterol in the membrane by interaction with another er membrane protein, called sCap (sreBp cleavage activation protein), which binds cholesterol. if the cholesterol binding site on sCap is empty (at low cholesterol concentrations), sCap changes conformation and is packaged together with sreBp into transport vesicles, which deliver their cargo to the Golgi apparatus, where two Golgi-resident proteases cleave sreBp to free its cytosolic domain from the membrane. The cytosolic domain then moves into the nucleus, where it binds to the promoters of genes that encode proteins involved in cholesterol biosynthesis and activates their transcription. in this way, more cholesterol is made


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Cell_Biology_Alberts. moves into the nucleus, where it binds to the promoters of genes that encode proteins involved in cholesterol biosynthesis and activates their transcription. in this way, more cholesterol is made when its concentration falls below a threshold.


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Cell_Biology_Alberts. Figure 12–17 The nuclear lamina. an electron micrograph of a portion of the nuclear lamina in a Xenopus oocyte prepared by freeze-drying and metal shadowing. The lamina is formed by a regular lattice of specialized intermediate filaments. lamins are only present in metazoan cells. other, yet-unknown proteins may serve similar functions in species that lack lamins. (Courtesy of Ueli aebi.) complex inner nuclear membrane outer nuclear membranenuclear envelope PHOSPHORYLATION OF LAMINS AND NPC PROTEINS DEPHOSPHORYLATION OF LAMINS FUSION OF ENVELOPED CHROMOSOMES FUSION OF NUCLEAR ENVELOPE FRAGMENTS nuclear envelope fragmentduplicated chromosome daughter chromosome phosphorylated lamins INTERPHASE NUCLEUS EARLY TELOPHASE PROPHASE LATE TELOPHASE nuclear pore complex proteins P P P P P P P P P P P P P P P P Figure 12–18 The breakdown and re-formation of the nuclear envelope and lamina during mitosis. phosphorylation of the lamins triggers the disassembly to the chromosome surface, where they


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Cell_Biology_Alberts. P P P P P P Figure 12–18 The breakdown and re-formation of the nuclear envelope and lamina during mitosis. phosphorylation of the lamins triggers the disassembly to the chromosome surface, where they assemble into new NPCs. At the same time, inner nuclear membrane proteins and dephosphorylated lamins bind again to chromatin. ER membranes wrap around groups of chromosomes until they form a sealed nuclear envelope (Movie 12.2). During this process, the NPCs start actively re-importing proteins that contain nuclear localization signals. Because the nuclear envelope is initially closely applied to the surface of the chromosomes, the newly formed nucleus excludes all proteins except those initially bound to the mitotic chromosomes and those that are selectively imported through NPCs. In this way, all other large proteins, including ribosomes, are kept out of the newly assembled nucleus.


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Cell_Biology_Alberts. As we discuss in Chapter 17, the cloud of Ran-GTP surrounding chromatin is also important in assembling the mitotic spindle in a dividing cell. The nuclear envelope consists of an inner and an outer nuclear membrane that are continuous with each other and with the ER membrane, and the space between the inner and outer nuclear membrane is continuous with the ER lumen. RNA molecules, which are made in the nucleus, and ribosomal subunits, which are assembled there, are exported to the cytosol; in contrast, all the proteins that function in the nucleus are synthesized in the cytosol and are then imported. The extensive traffic of materials between the nucleus and cytosol occurs through nuclear pore complexes (NPCs), which provide a direct passageway across the nuclear envelope. Small molecules diffuse passively through the NPCs, but large macromolecules have to be actively transported.


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Cell_Biology_Alberts. Proteins containing nuclear localization signals are actively transported into the nucleus through NPCs, while proteins containing nuclear export signals are transported out of the nucleus to the cytosol. Some proteins, including the nuclear of the nuclear lamina, which initiates the nuclear envelope to break up. Dephosphorylation of the lamins reverses the process. an analogous phosphorylation and dephosphorylation cycle occurs for some nucleoporins and proteins of the inner nuclear membrane, and some of these dephosphorylations are also involved in the reassembly process. as indicated, the nuclear envelope initially re-forms around individual decondensing daughter chromosomes. eventually, as decondensation progresses, these structures fuse to form a single complete nucleus. mitotic breakdown of the nuclear envelope occurs in all metazoan cells. however, in many other species, such as yeasts, the nuclear envelope remains intact during mitosis, and the nucleus divides by fission.


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Cell_Biology_Alberts. import and export receptors, continually shuttle between the cytosol and nucleus. The monomeric GTPase Ran provides both the free energy and the directionality for nuclear transport. Cells regulate the transport of nuclear proteins and RNA molecules through the NPCs by controlling the access of these molecules to the transport machinery. Newly transcribed messenger RNA and ribosomal RNA are exported from the nucleus as parts of large ribonucleoprotein complexes. Because nuclear localization signals are not removed, nuclear proteins can be imported repeatedly, as is required each time that the nucleus reassembles after mitosis. The TransporT of proTeins inTo miToChonDria anD ChloroplasTs


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Cell_Biology_Alberts. The TransporT of proTeins inTo miToChonDria anD ChloroplasTs Mitochondria and chloroplasts (a specialized form of plastids in green algae and plant cells) are double-membrane-enclosed organelles. They specialize in ATP synthesis, using energy derived from electron transport and oxidative phosphorylation in mitochondria and from photosynthesis in chloroplasts (discussed in Chapter 14). Although both organelles contain their own DNA, ribosomes, and other components required for protein synthesis, most of their proteins are encoded in the cell nucleus and imported from the cytosol. Each imported protein must reach the particular organelle subcompartment in which it functions.


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Cell_Biology_Alberts. There are different subcompartments in mitochondria (Figure 12–19A): the internal matrix space and the intermembrane space, which is continuous with the cristae space. These compartments are formed by the two concentric mitochondrial membranes: the inner membrane, which encloses the matrix space and forms extensive invaginations called cristae, and the outer membrane, which is in contact with the cytosol. Protein complexes provide boundaries at the junctions where the cristae invaginate and divide the inner membrane into two domains: one inner membrane domain surrounds the cristae space, and the other domain abuts the outer membrane. Chloroplasts also have an outer and inner membrane, which enclose an intermembrane space, and the stroma, which is the chloroplast equivalent of the mitochondrial matrix space (Figure 12–19B). They have an additional subcompartment, the thylakoid space, which is surrounded by the thylakoid membrane. The thylakoid membrane derives from the inner membrane


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Cell_Biology_Alberts. matrix space (Figure 12–19B). They have an additional subcompartment, the thylakoid space, which is surrounded by the thylakoid membrane. The thylakoid membrane derives from the inner membrane during plastid development and is pinched off to become discontinuous with it. Each of the subcompartments in mitochondria and chloroplasts contains a distinct set of proteins.


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Cell_Biology_Alberts. New mitochondria and chloroplasts are produced by the growth of preexisting organelles, followed by fission (discussed in Chapter 14). The growth depends mainly on the import of proteins from the cytosol. The imported proteins must be transported across a number of membranes in succession and end up in the appropriate place. The process of protein movement across membranes is called protein translocation. This section explains how it occurs. Figure 12–19 The subcompartments of mitochondria and chloroplasts. in contrast to the cristae of mitochondria (a), the thylakoids of chloroplasts (B) are not connected to the inner membrane and therefore form a sealed compartment with a separate internal space.


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Cell_Biology_Alberts. Proteins imported into mitochondria are usually taken up from the cytosol within seconds or minutes of their release from ribosomes. Thus, in contrast to protein translocation into the ER, which often takes place simultaneously with translation by a ribosome docked on the rough ER membrane (described later), mitochondrial proteins are first fully synthesized as mitochondrial precursor proteins in the cytosol and then translocated into mitochondria by a post-translational mechanism. One or more signal sequences direct all mitochondrial precursor proteins to their appropriate mitochondrial subcompartment. Many proteins entering the matrix space contain a signal sequence at their N-terminus that a signal peptidase rapidly removes after import. Other imported proteins, including all outer membrane and many inner membrane and intermembrane space proteins, have internal signal sequences that are not removed. The signal sequences are both necessary and sufficient for the import and correct


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Cell_Biology_Alberts. and many inner membrane and intermembrane space proteins, have internal signal sequences that are not removed. The signal sequences are both necessary and sufficient for the import and correct localization of the proteins: when genetic engineering techniques are used to link these signals to a cytosolic protein, the signals direct the protein to the correct mitochondrial subcompartment.


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Cell_Biology_Alberts. The signal sequences that direct precursor proteins into the mitochondrial matrix space are best understood. They all form an amphiphilic α helix, in which positively charged residues cluster on one side of the helix, while uncharged hydrophobic residues cluster on the opposite side. Specific receptor proteins that initiate protein translocation recognize this configuration rather than the precise amino acid sequence of the signal sequence (Figure 12–20). Multisubunit protein complexes that function as protein translocators mediate protein movement across mitochondrial membranes. The TOM complex transfers proteins across the outer membrane, and two TIM complexes (TIM23 and TIM22) transfer proteins across the inner membrane (Figure 12–21). These complexes contain some components that act as receptors for mitochondrial precursor proteins, and other components that form the translocation channels.


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Cell_Biology_Alberts. The TOM complex is required for the import of all nucleus-encoded mitochondrial proteins. It initially transports their signal sequences into the intermembrane space and helps to insert transmembrane proteins into the outer membrane. β-barrel proteins, which are particularly abundant in the outer membrane, are then passed on to an additional translocator, the SAM complex, which helps them to fold properly in the outer membrane. The TIM23 complex transports some soluble proteins into the matrix space and helps to insert transmembrane proteins into the inner membrane. The TIM22 complex mediates the insertion of a subclass of inner membrane proteins, including the transporter that moves ADP, ATP, and phosphate in and out of mitochondria. Yet another protein translocator in the inner mitochondrial membrane, the OXA complex, mediates the insertion of Figure 12–20 A signal sequence for mitochondrial protein import.


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Cell_Biology_Alberts. Cytochrome oxidase is a large multiprotein complex located in the inner mitochondrial membrane, where it functions as the terminal enzyme in the electron-transport chain (discussed in Chapter 14). (a) The first 18 amino acids of the precursor to subunit iV of this enzyme serve as a signal sequence for import of the subunit into the mitochondrion. (B) When the signal sequence is folded as an α helix, the positively charged amino acids (red) are clustered on one face of the helix, while the nonpolar ones (green) are clustered primarily on the opposite face. Uncharged polar amino acids are shaded orange; nitrogen atoms on the side chains of arg and Gln are colored blue. signal sequences that direct proteins into the matrix space always have the potential to form such an amphiphilic α helix, which is recognized by specific receptor proteins on the mitochondrial surface. (C) The structure of a signal sequence (of alcohol dehydrogenase, another mitochondrial matrix enzyme), bound to an


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Cell_Biology_Alberts. which is recognized by specific receptor proteins on the mitochondrial surface. (C) The structure of a signal sequence (of alcohol dehydrogenase, another mitochondrial matrix enzyme), bound to an import receptor (gray), as determined by nuclear magnetic resonance. The amphiphilic α helix binds with its hydrophobic face to a hydrophobic groove in the receptor (pDB code: 1om2).


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Cell_Biology_Alberts. 660 Chapter 12: intracellular Compartments and protein sorting those inner membrane proteins that are synthesized within mitochondria. It also helps to insert some imported inner membrane proteins that are initially transported into the matrix space by the other complexes. mitochondrial precursor proteins are imported as Unfolded polypeptide Chains We have learned almost everything we know about the molecular mechanism of protein import into mitochondria from analyses of cell-free, reconstituted trans-location systems, in which purified mitochondria in a test tube import radiolabeled mitochondrial precursor proteins. By changing the conditions in the test tube, it is possible to establish the biochemical requirements for the import.


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Cell_Biology_Alberts. Mitochondrial precursor proteins do not fold into their native structures after they are synthesized; instead, they remain unfolded in the cytosol through interactions with other proteins. Some of these interacting proteins are general chaperone proteins of the hsp70 family (discussed in Chapter 6), whereas others are dedicated to mitochondrial precursor proteins and bind directly to their signal sequences. All the interacting proteins help to prevent the precursor proteins from aggregating or folding up spontaneously before they engage with the TOM complex in the outer mitochondrial membrane. As a first step in the import process, the import receptors of the TOM complex bind the signal sequence of the mitochondrial precursor protein. The interacting proteins are then stripped off, and the unfolded polypeptide chain is fed—signal sequence first—into the trans-location channel.


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Cell_Biology_Alberts. In principle, a protein could reach the mitochondrial matrix space by either crossing the two membranes all at once or crossing one at a time. One can distinguish between these possibilities by cooling a cell-free mitochondrial import system to arrest the proteins at an intermediate step in the translocation process. The result is that the arrested proteins no longer contain their N-terminal signal sequence, indicating that the N-terminus must be in the matrix space where the signal peptidase is located, but the bulk of the protein can still be attacked from outside the mitochondria by externally added proteolytic enzymes. Clearly, the precursor proteins can pass through both mitochondrial membranes at once to enter the matrix space (Figure 12–22). The TOM complex first transports the signal sequence across the outer membrane to the intermembrane space, where it binds to a TIM complex, opening the channel in the complex. The polypeptide chain is then either translocated into the


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Cell_Biology_Alberts. signal sequence across the outer membrane to the intermembrane space, where it binds to a TIM complex, opening the channel in the complex. The polypeptide chain is then either translocated into the matrix space or inserted into the inner membrane.


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Cell_Biology_Alberts. Figure 12–21 The protein translocators in the mitochondrial membranes. The Tom, Tim, sam, and oXa complexes are multimeric membrane protein assemblies that catalyze protein translocation across mitochondrial membranes. The protein components of the Tim22 and Tim23 complexes that line the import channel are structurally related, suggesting a common evolutionary origin of both Tim complexes. on the matrix side, the Tim23 complex is bound to a multimeric protein complex containing mitochondrial hsp70, which acts as an import aTpase, using aTp hydrolysis to pull proteins through the pore. in animal cells, subtle variations exist in the subunit composition of the translocator complexes to adapt the mitochondrial import machinery to the particular needs of specialized cell types. sam = sorting and assembly machinery; oXa = cytochrome oXidase activity; Tim = Translocator of the inner mitochondrial membrane; Tom = Translocator of the outer membrane.


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Cell_Biology_Alberts. mitochondria. The n-terminal signal sequence of the mitochondrial precursor protein is recognized by receptors of the Tom complex. The protein is then translocated through the Tim23 complex so that it transiently spans both mitochondrial membranes (Movie 12.3). The signal sequence is cleaved off by a signal peptidase in the matrix space to form the mature protein. The free signal sequence is then rapidly degraded (not shown). Although the TOM and TIM complexes usually work together to translocate precursor proteins across both membranes at the same time, they can work independently. In isolated outer membranes, for example, the TOM complex can translocate the signal sequence of precursor proteins across the membrane. Similarly, if the outer membrane is experimentally disrupted in isolated mitochondria, the exposed TIM23 complex can efficiently import precursor proteins into the matrix space. aTp hydrolysis and a membrane potential Drive protein import into the matrix space


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Cell_Biology_Alberts. aTp hydrolysis and a membrane potential Drive protein import into the matrix space Directional transport requires energy, which in most biological systems is supplied by ATP hydrolysis. ATP hydrolysis fuels mitochondrial protein import at two discrete sites, one outside the mitochondria and one in the matrix space. In addition, protein import requires another energy source, which is the membrane potential across the inner mitochondrial membrane (Figure 12–23). The first requirement for energy occurs at the initial stage of the translocation process, when the unfolded precursor protein, associated with chaperone proteins, interacts with the import receptors of the TOM complex. As discussed in Chapter 6, the binding and release of newly synthesized polypeptides from the chaperone proteins requires ATP hydrolysis.


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Cell_Biology_Alberts. Figure 12–23 The role of energy in protein import into the mitochondrial matrix space. (1) Bound cytosolic hsp70 chaperone is released from the precursor protein in a step that depends on aTp hydrolysis. after initial insertion of the signal sequence and of adjacent portions of the polypeptide chain into the Tom complex translocation channel, the signal sequence interacts with a Tim complex. (2) The signal sequence is then translocated into the matrix space in a process that requires the energy in the membrane potential across the inner membrane. (3) Mitochondrial hsp70, which is part of an import aTpase complex, binds to regions of the polypeptide chain as they become exposed in the matrix space, pulling the protein through the translocation channel, using the energy of aTp hydrolysis. 662 Chapter 12: intracellular Compartments and protein sorting


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Cell_Biology_Alberts. 662 Chapter 12: intracellular Compartments and protein sorting Once the signal sequence has passed through the TOM complex and is bound to a TIM complex, further translocation through the TIM translocation channel requires the membrane potential, which is the electrical component of the electrochemical H+ gradient across the inner membrane (see Figure 11–4). Pumping of H+ from the matrix space to the intermembrane space, driven by electron transport processes in the inner membrane (discussed in Chapter 14), maintains the electrochemical gradient. The energy in the electrochemical H+ gradient across the inner membrane therefore not only powers most of the cell’s ATP synthesis, but it also drives the translocation of the positively charged signal sequences through the TIM complexes by electrophoresis.


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Cell_Biology_Alberts. Mitochondrial hsp70 also plays a crucial part in the import process. Mitochondria containing mutant forms of the protein fail to import precursor proteins. The mitochondrial hsp70 is part of a multisubunit protein assembly that is bound to the matrix side of the TIM23 complex and acts as a motor to pull the precursor protein into the matrix space. Like its cytosolic cousin, mitochondrial hsp70 has a high affinity for unfolded polypeptide chains, and it binds tightly to an imported protein chain as soon as the chain emerges from the TIM translocator in the matrix space. The hsp70 then undergoes a conformational change and releases the protein chain in an ATP-dependent step, exerting a ratcheting/pulling force on the protein being imported. This energy-driven cycle of binding and subsequent release provides the final driving force needed to complete protein import after a protein has initially inserted into the TIM23 complex (see Figure 12–23).


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Cell_Biology_Alberts. After the initial interaction with mitochondrial hsp70, many imported matrix proteins are passed on to another chaperone protein, mitochondrial hsp60. As discussed in Chapter 6, hsp60 helps the unfolded polypeptide chain to fold by binding and releasing it through cycles of ATP hydrolysis. Bacteria and mitochondria Use similar mechanisms to insert porins into their outer membrane


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Cell_Biology_Alberts. Bacteria and mitochondria Use similar mechanisms to insert porins into their outer membrane The outer mitochondrial membrane, like the outer membrane of Gram-negative bacteria (see Figure 11–17), contains abundant pore-forming β-barrel proteins called porins, and it is thus freely permeable to inorganic ions and metabolites (but not to most proteins). In contrast to other outer membrane proteins, which are anchored in the membrane through transmembrane α-helical regions, the TOM complex cannot integrate porins into the lipid bilayer. Instead, porins are first transported unfolded into the intermembrane space, where they transiently bind specialized chaperone proteins, which keep the porins from aggregating (Figure 12–24A). They then bind to the SAM complex in the outer membrane, which both inserts them into the outer membrane and helps them fold properly.


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Cell_Biology_Alberts. One of the central subunits of the SAM complex is homologous to a bacterial outer membrane protein that helps insert β-barrel proteins into the bacterial outer Figure 12–24 Integration of porins into the outer mitochondrial and bacterial membranes. (a) after translocation through the Tom complex in the outer mitochondrial membrane, β-barrel proteins bind to chaperones in the intermembrane space. The sam complex then inserts the unfolded polypeptide chain into the outer membrane and helps the chain fold. (B) a structurally related Bam complex in the outer membrane of Gram-negative bacteria catalyzes β-barrel protein insertion and folding (see figure 11–17). membrane from the periplasmic space (the equivalent of the intermembrane space in mitochondria) (Figure 12–24B). This conserved pathway for inserting β-barrel proteins further underscores the endosymbiotic origin of mitochondria. Transport into the inner mitochondrial membrane and intermembrane space occurs Via several routes


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Cell_Biology_Alberts. Transport into the inner mitochondrial membrane and intermembrane space occurs Via several routes The same mechanism that transports proteins into the matrix space using the TOM and TIM23 translocators (see Figure 12–22) also mediates the initial translocation of many proteins that are destined for the inner mitochondrial membrane or the intermembrane space. In the most common translocation route, only the N-terminal signal sequence of the transported protein actually enters the matrix space (Figure 12–25A). A hydrophobic amino acid sequence, strategically placed after the N-terminal signal sequence, acts as a stop-transfer sequence, preventing protease intermembrane reduced protein to TRANSLOCATION ACROSScleavage space protein be imported SH SH SH SHSHSH S S S SH S S S S S SH RECOGNITION AND OXIDATION BY Mia40 REOXIDATION BY RESPIRATORY CHAIN OUTER MEMBRANE REDUCTION OF Mia40, OXIDATION OF IMPORTED PROTEINMia40 TOM complex


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Cell_Biology_Alberts. Figure 12–25 Protein import from the cytosol into the inner mitochondrial membrane and intermembrane space. (a) The n-terminal signal sequence (red) initiates import into the matrix space (see figure 12–22). a hydrophobic sequence (blue) that follows the matrix-targeting signal sequence binds to the Tim23 translocator (orange) in the inner membrane and stops translocation. The remainder of the protein is then pulled into the intermembrane space through the Tom translocator in the outer membrane, and the hydrophobic sequence is released into the inner membrane anchoring the protein there. (B) a second route for protein integration into the inner membrane first delivers the protein completely into the matrix space. Cleavage of the signal sequence (red) used for the initial translocation unmasks an adjacent hydrophobic signal sequence (blue) at the new n-terminus. This signal then directs the protein into the inner membrane, using the same oXa-dependent pathway that inserts proteins that


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Cell_Biology_Alberts. an adjacent hydrophobic signal sequence (blue) at the new n-terminus. This signal then directs the protein into the inner membrane, using the same oXa-dependent pathway that inserts proteins that are encoded by the mitochondrial genome and translated in the matrix space. (C) some soluble proteins of the intermembrane space also use the pathways shown in (a) and (B) before they are released into the intermembrane space by a second signal peptidase, which has its active site in the intermembrane space and removes the hydrophobic signal sequence. (D) some soluble intermembrane-space proteins become oxidized by the mia40 protein (mia = mitochondrial intermembrane space assembly) during import. mia40 forms a covalent intermediate through an intermolecular disulfide bond, which helps pull the transported protein through the Tom complex. mia40 becomes reduced in the process, and then is reoxidized by the electron transport chain, so that it can catalyze the next round of import. (e)


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Cell_Biology_Alberts. the transported protein through the Tom complex. mia40 becomes reduced in the process, and then is reoxidized by the electron transport chain, so that it can catalyze the next round of import. (e) multipass inner membrane proteins that function as metabolite transporters contain internal signal sequences and snake through the Tom complex as a loop. They then bind to the chaperones in the intermembrane space, which guide the proteins to the Tim22 complex. The Tim22 complex is specialized for the insertion of multipass inner membrane proteins.


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Cell_Biology_Alberts. further translocation across the inner membrane. The remainder of the protein then crosses the outer membrane through the TOM complex into the intermembrane space; the signal sequence is cleaved off in the matrix, and the hydrophobic sequence, released from TIM23, remains anchored in the inner membrane.


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Cell_Biology_Alberts. In another transport route to the inner membrane or intermembrane space, the TIM23 complex initially translocates the entire protein into the matrix space (Figure 12–25B). A matrix signal peptidase then removes the N-terminal signal sequence, exposing a hydrophobic sequence at the new N-terminus. This signal sequence guides the protein to the OXA complex, which inserts the protein into the inner membrane. As mentioned earlier, the OXA complex is primarily used to insert proteins that are encoded and translated in the mitochondrion into the inner membrane, and only a few imported proteins use this pathway. Translocators that are closely related to the OXA complex are found in the plasma membrane of bacteria and in the thylakoid membrane of chloroplasts, where they insert membrane proteins by a similar mechanism.


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Cell_Biology_Alberts. Many proteins that use these pathways to the inner membrane remain anchored there through their hydrophobic signal sequence (see Figure 12–25A,B). Others, however, are released into the intermembrane space by a protease that removes the membrane anchor (Figure 12–25C). Many of these cleaved proteins remain attached to the outer surface of the inner membrane as peripheral subunits of protein complexes that also contain transmembrane proteins.


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Cell_Biology_Alberts. Certain intermembrane-space proteins that contain cysteine motifs are imported by a yet different route. These proteins form a transient covalent disulfide bond to the Mia40 protein (Figure 12–25D). The imported proteins are then released in an oxidized form containing intrachain disulfide bonds. Mia40 becomes reduced in the process, and is then reoxidized by passing electrons to the electron transport chain in the inner mitochondrial membrane. In this way, the energy stored in the redox potential in the mitochondrial electron transport chain is tapped to drive protein import.


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Cell_Biology_Alberts. Mitochondria are the principal sites of ATP synthesis in the cell, but they also contain many metabolic enzymes, such as those of the citric acid cycle. Thus, in addition to proteins, mitochondria must also transport small metabolites across their membranes. While the outer membrane contains porins, which make the membrane freely permeable to such small molecules, the inner membrane does not. Instead, a family of metabolite-specific transporters transfers a vast number of small molecules across the inner membrane. In yeast cells, these transporters comprise a family of 35 different proteins, the most abundant of which transport ATP, ADP, and phosphate. These are multipass transmembrane proteins, which do not have cleavable signal sequences at their N-termini but instead contain internal signal sequences. They cross the TOM complex in the outer membrane, and intermembrane-space chaperones guide them to the TIM22 complex, which inserts them into the inner membrane by a process that


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Cell_Biology_Alberts. signal sequences. They cross the TOM complex in the outer membrane, and intermembrane-space chaperones guide them to the TIM22 complex, which inserts them into the inner membrane by a process that requires the membrane potential, but not mitochondrial hsp70 or ATP (Figure 12–25E). An energetically favorable partitioning of the hydrophobic transmembrane regions into the inner membrane is likely to drive this process.


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Cell_Biology_Alberts. Two signal sequences Direct proteins to the Thylakoid membrane in Chloroplasts Protein transport into chloroplasts resembles transport into mitochondria. Both processes occur post-translationally, use separate translocation complexes in each membrane, require energy, and use amphiphilic N-terminal signal sequences that are removed after use. With the exception of some of the chaperone molecules, however, the protein components that form the translocation complexes differ. Moreover, whereas mitochondria harness the electrochemical H+ gradient across their inner membrane to drive transport, chloroplasts, which have an electrochemical H+ gradient across their thylakoid membrane but not their inner membrane, use GTP and ATP hydrolysis to power import across their double membrane. The functional similarities may thus result from convergent evolution, reflecting the common requirements for translocation across a double membrane.


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Cell_Biology_Alberts. Although the signal sequences for import into chloroplasts superficially resemble those for import into mitochondria, the same plant cells have both mitochondria and chloroplasts, so proteins must partition appropriately between the two organelles. In plants, for example, a bacterial enzyme can be directed specifically to mitochondria if it is experimentally joined to an N-terminal signal sequence of a mitochondrial protein; the same enzyme joined to an N-terminal signal sequence of a chloroplast protein ends up in chloroplasts. Thus, the import receptors on each organelle distinguish between the different signal sequences.


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Cell_Biology_Alberts. Chloroplasts have an extra membrane-enclosed compartment, the thylakoid. Many chloroplast proteins, including the protein subunits of the photosynthetic system and of the ATP synthase (discussed in Chapter 14), are located in the thylakoid membrane. Like the precursors of some mitochondrial proteins, the precursors of these proteins are translocated from the cytosol to their final destination in two steps. First, they pass across the double membrane into the matrix space (called the stroma in chloroplasts), and then they either integrate into the thylakoid membrane or translocate into the thylakoid space (Figure 12–26A). The precursors of these proteins have a hydrophobic thylakoid signal sequence following the N-terminal chloroplast signal sequence. After the N-terminal signal sequence has been used to import the protein into the stroma, a stromal signal peptidase removes it, unmasking the thylakoid signal sequence that initiates transport


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Cell_Biology_Alberts. Figure 12–26 Translocation of chloroplast precursor proteins into the thylakoid space. (a) The precursor protein contains an n-terminal chloroplast signal sequence (red), followed immediately by a thylakoid signal sequence (brown). The chloroplast signal sequence initiates translocation into the stroma by a mechanism similar to that used for the translocation of mitochondrial precursor proteins into the matrix space, although the translocator complexes, ToC and TiC, are different. The signal sequence is then cleaved off, unmasking the thylakoid signal sequence, which initiates translocation across the thylakoid membrane.


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Cell_Biology_Alberts. (B) Translocation into the thylakoid space or thylakoid membrane can occur by any one of at least four routes: (1) a Sec pathway, so called because it uses components that are homologs of sec proteins, which mediate protein translocation across the bacterial plasma membrane (discussed later); (2) an SRP-like pathway, so called because it uses a chloroplast homolog of the signal-recognition particle, or srp (discussed later); (3) a TAT (twin arginine translocation) pathway, so called because two arginines are critical in the signal sequences that direct proteins into this pathway, which depends on the h+ gradient across the thylakoid membrane; and (4) a spontaneous insertion pathway that seems not to require any protein translocator. across the thylakoid membrane. There are at least four routes by which proteins cross or become integrated into the thylakoid membrane, distinguished by their need for different stromal chaperones and energy sources (Figure 12–26B).


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Cell_Biology_Alberts. Although mitochondria and chloroplasts have their own genetic systems, they produce only a small proportion of their own proteins. Instead, the two organelles import most of their proteins from the cytosol, using similar mechanisms. In both cases, proteins are transported in an unfolded state across both outer and inner membranes simultaneously into the matrix space or stroma. Both ATP hydrolysis and a membrane potential across the inner membrane drive translocation into mitochondria, whereas GTP and ATP hydrolysis drive translocation into chloroplasts. Chaperone proteins of the cytosolic hsp70 family maintain the precursor proteins in an unfolded state, and a second set of hsp70 proteins in the matrix space or stroma pulls the polypeptide chain into the organelle. Only proteins that contain a specific signal sequence are translocated. The signal sequence can either be located at the N-terminus and cleaved off after import or be internal and retained. Transport into the inner membrane


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Cell_Biology_Alberts. a specific signal sequence are translocated. The signal sequence can either be located at the N-terminus and cleaved off after import or be internal and retained. Transport into the inner membrane sometimes uses a second, hydrophobic signal sequence that is unmasked when the first signal sequence is removed. In chloroplasts, import from the stroma into the thylakoid can occur by several routes, distinguished by the chaperones and energy source used.


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Cell_Biology_Alberts. Peroxisomes differ from mitochondria and chloroplasts in many ways. Most notably, they are surrounded by only a single membrane, and they do not contain DNA or ribosomes. Thus, because peroxisomes lack a genome, all of their proteins are encoded in the nucleus. Peroxisomes acquire most of these proteins by selective import from the cytosol, although some of them enter the peroxisome membrane via the ER. Because we do not discuss peroxisomes elsewhere, we shall digress to consider some of the functions of this diverse family of organelles, before discussing their biosynthesis. Virtually all eukaryotic cells have peroxisomes. They contain oxidative enzymes, such as catalase and urate oxidase, at such high concentrations that, in some cells, the peroxisomes stand out in electron micrographs because of the presence of a crystalloid protein core (Figure 12–27).


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Cell_Biology_Alberts. Like mitochondria, peroxisomes are major sites of oxygen utilization. One hypothesis is that peroxisomes are a vestige of an ancient organelle that performed all the oxygen metabolism in the primitive ancestors of eukaryotic cells. When the oxygen produced by photosynthetic bacteria first accumulated in the atmosphere, it would have been highly toxic to most cells. Peroxisomes might have lowered the intracellular concentration of oxygen, while also exploiting its chemical reactivity to perform useful oxidation reactions. According to this view, the later development of mitochondria rendered peroxisomes largely obsolete because many of the same biochemical reactions—which had formerly been carried out in peroxisomes without producing energy—were now coupled to ATP formation by means of oxidative phosphorylation. The oxidation reactions performed by peroxisomes in present-day cells could therefore partly be those whose functions were not taken over by mitochondria.


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Cell_Biology_Alberts. peroxisomes Use molecular oxygen and hydrogen peroxide to perform oxidation reactions Peroxisomes are so named because they usually contain one or more enzymes that use molecular oxygen to remove hydrogen atoms from specific organic substrates (designated here as R) in an oxidation reaction that produces hydrogen peroxide (H2O2): Catalase uses the H2O2 generated by other enzymes in the organelle to oxidize a variety of other substrates—including formic acid, formaldehyde, and alcohol— by the “peroxidation” reaction: H2O2 + R′H2 → R′ + 2H2O. This type of oxidation reaction is particularly important in liver and kidney cells, where the peroxisomes detoxify various harmful molecules that enter the bloodstream. About 25% of the ethanol we drink is oxidized to acetaldehyde in this way. In addition, when excess H2O2 accumulates in the cell, catalase converts it to H2O through the reaction


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Cell_Biology_Alberts. A major function of the oxidation reactions performed in peroxisomes is the breakdown of fatty acid molecules. The process, called β oxidation, shortens the alkyl chains of fatty acids sequentially in blocks of two carbon atoms at a time, thereby converting the fatty acids to acetyl CoA. The peroxisomes then export the acetyl CoA to the cytosol for use in biosynthetic reactions. In mammalian cells, β oxidation occurs in both mitochondria and peroxisomes; in yeast and plant cells, however, this essential reaction occurs exclusively in peroxisomes. An essential biosynthetic function of animal peroxisomes is to catalyze the first reactions in the formation of plasmalogens, which are the most abundant class of phospholipids in myelin (Figure 12–28). Plasmalogen deficiencies cause profound abnormalities in the myelination of nerve-cell axons, which is one reason why many peroxisomal disorders lead to neurological disease.


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Cell_Biology_Alberts. Peroxisomes are unusually diverse organelles, and even in the various cell types of a single organism they may contain different sets of enzymes. They also adapt remarkably to changing conditions. Yeasts grown on sugar, for example, have few small peroxisomes. But when some yeasts are grown on methanol, numerous large peroxisomes are formed that oxidize methanol; and when grown on fatty acids, they develop numerous large peroxisomes that break down fatty acids to acetyl CoA by β oxidation.


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Cell_Biology_Alberts. Peroxisomes are also important in plants. Two types of plant peroxisomes have been studied extensively. One is present in leaves, where it participates in photo-respiration (discussed in Chapter 14) (Figure 12–29A). The other type of peroxisome is present in germinating seeds, where it converts the fatty acids stored in seed lipids into the sugars needed for the growth of the young plant. Because this conversion of fats to sugars is accomplished by a series of reactions known as the glyoxylate cycle, these peroxisomes are also called glyoxysomes (Figure 12–29B). In the glyoxylate cycle, two molecules of acetyl CoA produced by fatty acid breakdown in the peroxisome are used to make succinic acid, which then leaves the peroxisome and is converted into glucose in the cytosol. The glyoxylate cycle does not occur in animal cells, and animals are therefore unable to convert the fatty acids in fats into carbohydrates. a short signal sequence Directs the import of proteins into peroxisomes


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Cell_Biology_Alberts. A specific sequence of three amino acids (Ser–Lys–Leu) located at the C-terminus of many peroxisomal proteins functions as an import signal (see Table 12–3, p. 648). Other peroxisomal proteins contain a signal sequence near the N-terminus. If either sequence is attached to a cytosolic protein, the protein is imported into peroxisomes. The import signals are first recognized by soluble receptor proteins in the cytosol. Numerous distinct proteins, called peroxins, participate in the import process, which is driven by ATP hydrolysis. A complex of at least six different peroxins forms a protein translocator in the peroxisome membrane. Even oligomeric proteins do not have to unfold to be imported. To allow the passage of such compactly folded cargo molecules, the pore formed by the transporter is thought to be dynamic in its dimensions, adapting in size to the particular cargo molecules to be transported. In this respect, the mechanism differs from that used by mitochondria and


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Cell_Biology_Alberts. transporter is thought to be dynamic in its dimensions, adapting in size to the particular cargo molecules to be transported. In this respect, the mechanism differs from that used by mitochondria and chloroplasts. One soluble import receptor, the peroxin Pex5 recognizes the C-terminal peroxisomal import signal. It accompanies its cargo all the way into peroxisomes and, after cargo release, cycles back to the cytosol. After


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Cell_Biology_Alberts. Figure 12–27 An electron micrograph of three peroxisomes in a rat liver cell. The paracrystalline, electron-dense inclusions are composed primarily of the enzyme urate oxidase. (Courtesy of Daniel s. friend.) Figure 12–28 The structure of a plasmalogen. plasmalogens are very abundant in the myelin sheaths that insulate the axons of nerve cells. They make up some 80–90% of the myelin membrane phospholipids. in addition to an ethanolamine head group and a long-chain fatty acid attached to the same glycerol phosphate backbone used for phospholipids, plasmalogens contain an unusual fatty alcohol that is attached through an ether linkage (bottom left).


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Cell_Biology_Alberts. Figure 12–29 Electron micrographs of two types of peroxisomes found in plant cells. (a) a peroxisome with a paracrystalline core in a tobacco leaf mesophyll cell. its close association with chloroplasts is thought to facilitate the exchange of materials between these organelles during photorespiration. The vacuole in plant cells is equivalent to the lysosome in animal cells. (B) peroxisomes in a fat-storing cotyledon cell of a tomato seed 4 days after germination. here the peroxisomes (glyoxysomes) are associated with the lipid droplets that store fat, reflecting their central role in fat mobilization and gluconeogenesis during seed germination. (a, from s.e. frederick and e.h. newcomb, J. Cell Biol. 43:343–353, 1969. With permission from The rockefeller press; B, from W.p. Wergin, p.J. Gruber and e.h. newcomb, J. Ultrastruct. Res. 30:533–557, 1970. With permission from academic press.) delivering its cargo to the peroxisome lumen, Pex5 undergoes ubiquitylation. This modification is


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Cell_Biology_Alberts. Gruber and e.h. newcomb, J. Ultrastruct. Res. 30:533–557, 1970. With permission from academic press.) delivering its cargo to the peroxisome lumen, Pex5 undergoes ubiquitylation. This modification is required to release Pex5 back into the cytosol, where the ubiquitin is removed. An ATPase composed of Pex1 and Pex6 harnesses the energy of ATP hydrolysis to help release Pex5 from peroxisomes.


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Cell_Biology_Alberts. The importance of this import process and of peroxisomes is demonstrated by the inherited human disease Zellweger syndrome, in which a defect in importing proteins into peroxisomes leads to a profound peroxisomal deficiency. These individuals, whose cells contain “empty” peroxisomes, have severe abnormalities in their brain, liver, and kidneys, and they die soon after birth. A mutation in the gene encoding peroxin Pex5 causes one form of the disease. A defect in Pex7, the receptor for the N-terminal import signal, causes a milder peroxisomal disease. It has long been debated whether new peroxisomes arise from preexisting ones by organelle growth and fission—as mentioned earlier for mitochondria and plastids—or whether they derive as a specialized compartment from the endoplasmic reticulum (ER). Aspects of both views are true (Figure 12–30). Most peroxisomal membrane proteins are made in the cytosol and insert into the membrane of


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Cell_Biology_Alberts. Figure 12–30 A model that explains how peroxisomes proliferate and how new peroxisomes arise. peroxisomal precursor vesicles bud from the er. at least two peroxisomal membrane proteins, pex3 and pex15, follow this route. The machinery that drives the budding reaction and that selects only peroxisomal proteins for packaging into these vesicles depends on pex19 and other cytosolic proteins that are still unknown. peroxisomal precursor vesicles may then fuse with one another or with preexisting peroxisomes. The peroxisome membrane contains import receptors and protein translocators that are required for the import of peroxisomal proteins made on cytosolic ribosomes, including new copies of the import receptors and translocator components. presumably, the lipids required for growth are also imported, although some may derive directly from the er in the membrane of peroxisomal precursor vesicles.


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Cell_Biology_Alberts. preexisting peroxisomes, but others are first integrated into the ER membrane, where they are packaged into specialized peroxisomal precursor vesicles. New precursor vesicles may then fuse with one another and begin importing additional peroxisomal proteins, using their own protein import machinery to grow into mature peroxisomes, which can undergo cycles of growth and fission.


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Cell_Biology_Alberts. Peroxisomes are specialized for carrying out oxidation reactions using molecular oxygen. They generate hydrogen peroxide, which they employ for oxidative purposes—and contain catalase to destroy the excess. Like mitochondria and plastids, peroxisomes are self-replicating organelles. Because they do not contain DNA or ribosomes, however, all of their proteins are encoded in the cell nucleus. Some of these proteins are conveyed to peroxisomes via peroxisomal precursor vesicles that bud from the ER, but most are synthesized in the cytosol and directly imported. A specific sequence of three amino acids near the C-terminus of many of the latter proteins functions as a peroxisomal import signal. The mechanism of protein import differs from that of mitochondria and chloroplasts, in that even oligomeric proteins are imported from the cytosol without unfolding. The enDoplasmiC reTiCUlUm


Cell_Biology_Alberts

Cell_Biology_Alberts. The enDoplasmiC reTiCUlUm All eukaryotic cells have an endoplasmic reticulum (ER). Its membrane typically constitutes more than half of the total membrane of an average animal cell (see Table 12–2, p. 643). The ER is organized into a netlike labyrinth of branching tubules and flattened sacs that extends throughout the cytosol (Figure 12–31 and Movie 12.4). The tubules and sacs interconnect, and their membrane is continuous with the outer nuclear membrane; the compartment that they enclose therefore is also continuous with the space between the inner and outer nuclear membranes. Thus, the ER and nuclear membranes form a continuous sheet enclosing a single internal space, called the ER lumen or the ER cisternal space, which often occupies more than 10% of the total cell volume (see Table 12–1, p. 643).


Cell_Biology_Alberts

Cell_Biology_Alberts. As mentioned at the beginning of this chapter, the ER has a central role in both lipid and protein biosynthesis, and it also serves as an intracellular Ca2+ store that is used in many cell signaling responses (discussed in Chapter 15). The ER membrane is the site of production of all the transmembrane proteins and lipids for most of the cell’s organelles, including the ER itself, the Golgi apparatus, lysosomes, endosomes, secretory vesicles, and the plasma membrane. The ER membrane is also the site at which most of the lipids for mitochondrial and peroxisomal membranes are made. In addition, almost all of the proteins that will be secreted to the cell exterior—plus those destined for the lumen of the ER, Golgi apparatus, or lysosomes—are initially delivered to the ER lumen.


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Cell_Biology_Alberts. Figure 12–31 Fluorescent micrographs of the endoplasmic reticulum. (a) an animal cell in tissue culture that was genetically engineered to express an er membrane protein fused to a fluorescent protein. The er extends as a network of tubules and sheets throughout the entire cytosol, so that all regions of the cytosol are close to some portion of the er membrane. The outer nuclear membrane, which is continuous with the er, is also stained. (B) part of an er network in a living plant cell that was genetically engineered to express a fluorescent protein in the er. (a, courtesy of patrick Chitwood and Gia Voeltz; B, courtesy of petra Boevink and Chris hawes.) The er is structurally and functionally Diverse


Cell_Biology_Alberts

Cell_Biology_Alberts. The er is structurally and functionally Diverse While the various functions of the ER are essential to every cell, their relative importance varies greatly between individual cell types. To meet different functional demands, distinct regions of the ER become highly specialized. We observe such functional specialization as dramatic changes in ER structure, and different cell types can therefore possess characteristically different types of ER membrane. One of the most remarkable ER specializations is the rough ER.


Cell_Biology_Alberts

Cell_Biology_Alberts. Mammalian cells begin to import most proteins into the ER before complete synthesis of the polypeptide chain—that is, import is a co-translational process (Figure 12–32A). In contrast, the import of proteins into mitochondria, chloroplasts, nuclei, and peroxisomes is a post-translational process (Figure 12–32B). In co-translational transport, the ribosome that is synthesizing the protein is attached directly to the ER membrane, enabling one end of the protein to be translocated into the ER while the rest of the polypeptide chain is being synthesized. These membrane-bound ribosomes coat the surface of the ER, creating regions termed rough endoplasmic reticulum, or rough ER; regions of ER that lack bound ribosomes are called smooth endoplasmic reticulum, or smooth ER (Figure 12–33).


Cell_Biology_Alberts

Cell_Biology_Alberts. Most cells have scanty regions of smooth ER, and the ER is often partly smooth and partly rough. Areas of smooth ER from which transport vesicles carrying newly synthesized proteins and lipids bud off for transport to the Golgi apparatus are called transitional ER. In certain specialized cells, the smooth ER is abundant and has additional functions. It is prominent, for example, in cells that specialize in lipid metabolism, such as cells that synthesize steroid hormones from cholesterol; the expanded smooth ER accommodates the enzymes that make cholesterol and modify it to form the hormones (see Figure 12–33B).


Cell_Biology_Alberts

Cell_Biology_Alberts. The main cell type in the liver, the hepatocyte, also has a substantial amount of smooth ER. It is the principal site of production of lipoprotein particles, which carry lipids via the bloodstream to other parts of the body. The enzymes that synthesize the lipid components of the particles are located in the membrane of the smooth ER, which also contains enzymes that catalyze a series of reactions to detoxify both lipid-soluble drugs and various harmful compounds produced by metabolism. The most extensively studied of these detoxification reactions are carried out by the cytochrome P450 family of enzymes, which catalyze a series of reactions in which water-insoluble drugs or metabolites that would otherwise accumulate to toxic levels in cell membranes are rendered sufficiently water-soluble to leave the cell and be excreted in the urine. Because the rough ER alone cannot house enough of these and other necessary enzymes, a substantial portion of the membrane in a hepatocyte normally


Cell_Biology_Alberts

Cell_Biology_Alberts. to leave the cell and be excreted in the urine. Because the rough ER alone cannot house enough of these and other necessary enzymes, a substantial portion of the membrane in a hepatocyte normally consists of smooth ER (see Table 12–2).


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Cell_Biology_Alberts. Another crucially important function of the ER in most eukaryotic cells is to sequester Ca2+ from the cytosol. The release of Ca2+ into the cytosol from the ER, and its subsequent reuptake, occurs in many rapid responses to extracellular Figure 12–32 Co-translational and post-translational protein translocation. (a) ribosomes bind to the er membrane during co-translational translocation. (B) By contrast, cytosolic ribosomes complete the synthesis of a protein and release it prior to post-translational translocation. in both cases, the protein is directed to the er by an er signal sequence (red and orange). 200 nm 0.5 µm


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Cell_Biology_Alberts. 200 nm 0.5 µm Figure 12–33 The rough and smooth ER. (a) an electron micrograph of the rough er in a pancreatic exocrine cell that makes and secretes large amounts of digestive enzymes every day. The cytosol is filled with closely packed sheets of er membrane that is studded with ribosomes. at the top left is a portion of the nucleus and its nuclear envelope; note that the outer nuclear membrane, which is continuous with the er, is also studded with ribosomes. (B) abundant smooth er in a steroid-hormone-secreting cell. This electron micrograph is of a testosterone-secreting leydig cell in the human testis.


Cell_Biology_Alberts

Cell_Biology_Alberts. (C) a three-dimensional reconstruction of a region of smooth er and rough er in a liver cell. The rough er forms oriented stacks of flattened cisternae, each having a lumenal space 20–30 nm wide. The smooth er membrane is connected to these cisternae and forms a fine network of tubules 30–60 nm in diameter. The er lumen is colored green. (D) a tomographic reconstruction of a portion of the er network in a yeast cell. membrane-bound ribosomes (tiny dark spheres) are seen in both flat sheets and tubular regions of irregular diameter, demonstrating that the ribosomes bind to er membranes of different curvature in these cells. (a, courtesy of lelio orci; B, courtesy of Daniel s. friend; C, after r.V. Krstic´, Ultrastructure of the mammalian Cell. new York: springer-Verlag, 1979; D, from m. West et al., J. Cell Biol. 193:333–346, 2011. With permission from rockefeller University press.) signals, as discussed in Chapter 15. A Ca2+ pump transports Ca2+ from the cytosol into the ER lumen. A


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Cell_Biology_Alberts. et al., J. Cell Biol. 193:333–346, 2011. With permission from rockefeller University press.) signals, as discussed in Chapter 15. A Ca2+ pump transports Ca2+ from the cytosol into the ER lumen. A high concentration of Ca2+-binding proteins in the ER facilitates Ca2+ storage. In some cell types, and perhaps in most, specific regions of the ER are specialized for Ca2+ storage. Muscle cells have an abundant, modified smooth ER called the sarcoplasmic reticulum. The release and reuptake of Ca2+ by the sarcoplasmic reticulum trigger myofibril contraction and relaxation, respectively, during each round of muscle contraction (discussed in Chapter 16).


Cell_Biology_Alberts

Cell_Biology_Alberts. To study the functions and biochemistry of the ER, it is necessary to isolate it. This may seem to be a hopeless task because the ER is intricately interleaved with other components of the cytoplasm. Fortunately, when tissues or cells are disrupted by homogenization, the ER breaks into fragments, which reseal to form small (~100–200 nm in diameter) closed vesicles called microsomes. Microsomes are relatively easy to purify. To the biochemist, microsomes represent small authentic versions of the ER, still capable of protein translocation, protein glycosylation (discussed later), Ca2+ uptake and release, and lipid synthesis. Microsomes derived from rough ER are studded with ribosomes and are called rough microsomes. The ribosomes are always found on the outside surface, so the interior of the microsome is biochemically equivalent to the lumen of the ER (Figure 12–34A).


Cell_Biology_Alberts

Cell_Biology_Alberts. Many vesicles similar in size to rough microsomes, but lacking attached ribosomes, are also found in cell homogenates. Such smooth microsomes are derived in part from smooth portions of the ER and in part from vesiculated fragments of the plasma membrane, Golgi apparatus, endosomes, and mitochondria (the ratio depending on the tissue). Thus, whereas rough microsomes are clearly derived from rough portions of ER, it is not easy to separate smooth microsomes derived from different organelles. The smooth microsomes prepared from liver or muscle cells are an exception. Because of the unusually large quantities of smooth ER or sarcoplasmic reticulum, respectively, most of the smooth microsomes in homogenates of these tissues are derived from the smooth ER or sarcoplasmic reticulum. The ribosomes attached to rough microsomes make them more dense than smooth microsomes. As a result, we can use equilibrium centrifugation to separate the rough and smooth microsomes (Figure 12–34B). Microsomes


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Cell_Biology_Alberts. attached to rough microsomes make them more dense than smooth microsomes. As a result, we can use equilibrium centrifugation to separate the rough and smooth microsomes (Figure 12–34B). Microsomes have been invaluable in elucidating the molecular aspects of ER function, as we discuss next.


Cell_Biology_Alberts

Cell_Biology_Alberts. signal sequences Were first Discovered in proteins imported into the rough er The ER captures selected proteins from the cytosol as they are being synthesized. These proteins are of two types: transmembrane proteins, which are only partly translocated across the ER membrane and become embedded in it, and water-soluble proteins, which are fully translocated across the ER membrane and are released into the ER lumen. Some of the transmembrane proteins function in the ER, but many are destined to reside in the plasma membrane or the membrane of another organelle. The water-soluble proteins are destined either for secretion or for residence in the lumen of the ER or of another organelle. All of these proteins, regardless of their subsequent fate, are directed to the ER membrane by an ER signal sequence, which initiates their translocation by a common mechanism.


Cell_Biology_Alberts

Cell_Biology_Alberts. Signal sequences (and the signal sequence strategy of protein sorting) were first discovered in the early 1970s in secreted proteins that are translocated across the ER membrane as a first step toward their eventual discharge from the cell. In the key experiment, the mRNA encoding a secreted protein was translated by ribosomes in vitro. When microsomes were omitted from this cell-free system, the protein synthesized was slightly larger than the normal secreted protein. In the presence of microsomes derived from the rough ER, however, a protein of the correct size was produced. According to the signal hypothesis, the size difference reflects the initial presence of a signal sequence that directs the secreted protein


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 12–34 The isolation of purified rough and smooth microsomes from the ER. (a) a thin section electron micrograph of the purified rough er fraction shows an abundance of ribosome-studded vesicles. (B) When sedimented to equilibrium through a gradient of sucrose, the two types of microsomes separate from each other on the basis of their different densities. note that the smooth fraction will also contain non-er-derived material. (a, courtesy of George palade.) Figure 12–35 The signal hypothesis.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 12–35 The signal hypothesis. a simplified view of protein translocation across the er membrane, as originally proposed. When the er signal sequence emerges from the ribosome, it directs the ribosome to a translocator on the er membrane that forms a pore in the membrane through which the polypeptide is translocated. a signal peptidase is closely associated with the translocator and clips off the signal sequence during translation, and the mature protein is released into the lumen of the er immediately after its synthesis is completed. The translocator is closed until the ribosome has bound, so that the permeability barrier of the er membrane is maintained at all times.


Cell_Biology_Alberts

Cell_Biology_Alberts. to the ER membrane and is then cleaved off by a signal peptidase in the ER membrane before the polypeptide chain has been completed (Figure 12–35). Cell-free systems in which proteins are imported into microsomes have provided powerful procedures for identifying, purifying, and studying the various components of the molecular machinery responsible for the ER import process. a signal-recognition particle (srp) Directs the er signal sequence to a specific receptor in the rough er membrane


Cell_Biology_Alberts

Cell_Biology_Alberts. a signal-recognition particle (srp) Directs the er signal sequence to a specific receptor in the rough er membrane The ER signal sequence is guided to the ER membrane by at least two components: a signal-recognition particle (SRP), which cycles between the ER membrane and the cytosol and binds to the signal sequence, and an SRP receptor in the ER membrane. The SRP is a large complex; in animal cells, it consists of six different polypeptide chains bound to a single small RNA molecule. While the SRP and SRP receptor have fewer subunits in bacteria, homologs are present in all cells, indicating that this protein-targeting mechanism arose early in evolution and has been conserved.


Cell_Biology_Alberts

Cell_Biology_Alberts. ER signal sequences vary greatly in amino acid sequence, but each has eight or more nonpolar amino acids at its center (see Table 12–3, p. 648). How can the SRP bind specifically to so many different sequences? The answer has come from the crystal structure of the SRP protein, which shows that the signal-sequence-binding site is a large hydrophobic pocket lined by methionines. Because methionines have unbranched, flexible side chains, the pocket is sufficiently plastic to accommodate hydrophobic signal sequences of different sequences, sizes, and shapes.


Cell_Biology_Alberts

Cell_Biology_Alberts. The SRP is a rodlike structure, which wraps around the large ribosomal subunit, with one end binding to the ER signal sequence as it emerges from the ribosome as part of the newly made polypeptide chain; the other end blocks the elongation factor binding site at the interface between the large and small ribosomal subunits (Figure 12–36). This block halts protein synthesis as soon as the signal peptide has emerged from the ribosome. The transient pause presumably gives the ribosome enough time to bind to the ER membrane before completion of the polypeptide chain, thereby ensuring that the protein is not released into the cytosol. This safety


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Cell_Biology_Alberts. Figure 12–36 The signal-recognition particle (SRP). (a) a mammalian srp is a rodlike ribonucleoprotein complex containing six protein subunits (brown) and one rna molecule (blue). The srp rna forms a backbone that links the protein domain containing the signal-sequencebinding pocket to the domain responsible for pausing translation. Crystal structures of various srp pieces from different species are assembled here into a composite model to approximate the structure of a complete srp. (B) The three-dimensional outline of the srp bound to a ribosome was determined by cryoelectron microscopy. srp binds to the large ribosomal subunit so that its signal-sequence-binding pocket is positioned near the growing polypeptide chain exit site, and its translational pause domain is positioned at the interface between the ribosomal subunits, where it interferes with elongation factor binding. (C) as a signal sequence emerges from the ribosome and binds to the srp, a conformational change in the srp


Cell_Biology_Alberts

Cell_Biology_Alberts. between the ribosomal subunits, where it interferes with elongation factor binding. (C) as a signal sequence emerges from the ribosome and binds to the srp, a conformational change in the srp exposes a binding site for the srp receptor. (B, adapted from m. halic et al., Nature 427:808–814, 2004. With permission from macmillan publishers ltd.) device may be especially important for secreted and lysosomal hydrolases, which could wreak havoc in the cytosol; cells that secrete large amounts of hydrolases, however, take the added precaution of having high concentrations of hydrolase inhibitors in their cytosol. The pause also ensures that large portions of a protein that could fold into a compact structure are not made before reaching the translocator in the ER membrane. Thus, in contrast to the post-translational import of proteins into mitochondria and chloroplasts, chaperone proteins are not required to keep the protein unfolded.


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Cell_Biology_Alberts. When a signal sequence binds, SRP exposes a binding site for the SRP receptor (see Figure 12–36B,C), which is a transmembrane protein complex in the rough ER membrane. The binding of the SRP to its receptor brings the SRP–ribosome complex to an unoccupied protein translocator in the same membrane. The SRP and SRP receptor are then released, and the translocator transfers the growing polypeptide chain across the membrane (Figure 12–37). This co-translational transfer process creates two spatially separate populations of ribosomes in the cytosol. Membrane-bound ribosomes, attached to the


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 12–37 How ER signal sequences and SRP direct ribosomes to the ER membrane. The srp and its receptor act in concert. The srp binds to both the exposed er signal sequence and the ribosome, thereby inducing a pause in translation. The srp receptor in the er membrane, which in animal cells is composed of two different polypeptide chains, binds the srp–ribosome complex and directs it to the translocator. in a poorly understood reaction, the srp and srp receptor are then released, leaving the ribosome bound to the translocator in the er membrane. The translocator then inserts the polypeptide chain into the membrane and transfers it across the lipid bilayer. Because one of the srp proteins and both chains of the srp receptor contain GTp-binding domains, it is thought that conformational changes that occur during cycles of GTp binding and hydrolysis (discussed in Chapter 15) ensure that srp release occurs only after the ribosome has become properly engaged with the translocator in the


Cell_Biology_Alberts

Cell_Biology_Alberts. that occur during cycles of GTp binding and hydrolysis (discussed in Chapter 15) ensure that srp release occurs only after the ribosome has become properly engaged with the translocator in the er membrane. The translocator is closed until the ribosome has bound, so that the permeability barrier of the er membrane is maintained at all times.


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Cell_Biology_Alberts. cytosolic side of the ER membrane, are engaged in the synthesis of proteins that are being concurrently translocated into the ER. Free ribosomes, unattached to any membrane, synthesize all other proteins encoded by the nuclear genome. Membrane-bound and free ribosomes are structurally and functionally identical. They differ only in the proteins they are making at any given time.


Cell_Biology_Alberts

Cell_Biology_Alberts. Since many ribosomes can bind to a single mRNA molecule, a polyribosome is usually formed. If the mRNA encodes a protein with an ER signal sequence, the polyribosome becomes attached to the ER membrane, directed there by the signal sequences on multiple growing polypeptide chains. The individual ribosomes associated with such an mRNA molecule can return to the cytosol when they finish translation and intermix with the pool of free ribosomes. The mRNA itself, however, remains attached to the ER membrane by a changing population of ribosomes, each transiently held at the membrane by the translocator (Figure 12–38). The polypeptide Chain passes Through an aqueous Channel in the Translocator