extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

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Role of positive and negative cis-regulatory elements in the transcriptional activation of the lysozyme locus in developing macrophages of transgenic mice.

Expression of the chicken lysozyme locus in macrophages is regulated by at least six different positive and negative cis-regulatory elements. Chromatin of the chicken lysozyme locus is gradually reorganized during macrophage differentiation, indicating that each cis-regulatory element is activated at a different developmental stage. Irrespective of their differential developmental activation, individual cis-regulatory regions are capable of driving transcription of the lysozyme gene in mature macrophages of transgenic mice. In order to examine the role of different cis-regulatory regions in lysozyme locus activation, we analyzed the time course of transcriptional up-regulation of deletion mutants of the lysozyme locus in a new in vitro differentiation system based on enriched primary macrophage precursor cells from the bone marrow of transgenic mice. We show that constructs carrying cis-regulatory elements which are structurally reorganized early in development are also transcriptionally active at an early stage. A construct in which the early enhancer has been deleted shows a delay in transcriptional activation. The presence or absence of a negative regulatory element has no influence on the time course of transcriptional activation of the lysozyme locus.

Expression of the chicken lysozyme locus in macrophages is regulated by at least six different positive and negative cis-regulatory elements. Chromatin of the chicken lysozyme locus is gradually reorganized during macrophage differentiation, indicating that each cisregulatory element is activated at a different developmental stage. Irrespective of their differential developmental activation, individual cis-regulatory regions are capable of driving transcription of the lysozyme gene in mature macrophages of transgenic mice. In order to examine the role of different cis-regulatory regions in lysozyme locus activation, we analyzed the time course of transcriptional up-regulation of deletion mutants of the lysozyme locus in a new in vitro differentiation system based on enriched primary macrophage precursor cells from the bone marrow of transgenic mice. We show that constructs carrying cis-regulatory elements which are structurally reorganized early in development are also transcriptionally active at an early stage. A construct in which the early enhancer has been deleted shows a delay in transcriptional activation. The presence or absence of a negative regulatory element has no influence on the time course of transcriptional activation of the lysozyme locus.
Most gene loci examined so far are regulated by a variety of different cis-regulatory elements distributed over many kilobases of DNA. The presence of a DNase I-hypersensitive site (DHS) 1 in chromatin is the result of the assembly of transcription factor complexes; hence, a DHS in most cases indicates the presence of an active cis-regulatory element. Many gene loci exhibit changes in DHS patterns according to the developmental stage, indicating that along with cellular differentiation, individual cis-regulatory elements of a given gene locus display different activity patterns. It has been argued that a reorganization of chromatin at early stages of cellular differentiation may represent a priming step required for the assembly of an active transcription machinery at later stages of development (1)(2)(3)(4). However, the hierarchical relationship between the structural reorganization of cis-regulatory elements and their actual ability to stimulate mRNA synthesis is still unclear (1,2,(5)(6)(7). The dissection of the role of different cis-regulatory elements in the developmental control of gene locus activation requires their individual analysis in an experimental system where cell differentiation can be followed, thus enabling to link a stage-specific chromatin structure with the transcriptional activity of the gene.
We have been studying the chicken lysozyme locus as a marker for macrophage differentiation. Cis-regulatory elements regulating gene expression are located in the 5Ј-half of the gene locus. Transfection analysis revealed three enhancers located 6.1, 3.9, and 2.7 kb upstream of the transcriptional start site, a hormone responsive element at Ϫ1.9 kb, and a complex promoter (8 -14). 2 A negative regulatory element is located at Ϫ2.4 kb (Ϫ2.4-kb silencer), which has been implicated in the repression of the lysozyme locus in lysozyme nonexpressing tissues (11,15,16). The activity of each of these cis-regulatory elements is marked by the presence of DHS in chromatin (17)(18)(19)(20). In turn, reporter gene constructs carrying particular cis-regulatory elements are only active when transfected into cell types displaying a DHS at the position of the same element (13,14,19). The analysis of lysozyme chromatin structure in retrovirally transformed chicken cell lines resembling multipotent myeloid progenitor cells (21) and various macrophage maturation stages revealed that different sets of cis-regulatory elements are structurally reorganized at different developmental stages and thus are differentially active (20,22). Multipotent progenitor cells do not transcribe the lysozyme gene and display the chromatin structure of the inactive locus. At the myeloblast stage, DHSs are formed at the Ϫ6.1and the Ϫ3.9-kb (early) enhancers as well as at the promoter form. The silencer element is still active, as indicated by the presence of a DHS, whereas the (late) enhancer at Ϫ2.7 kb displays no DHS and thus is inactive at this differentiation stage. A low level of lysozyme mRNA can be measured. At later stages of differentiation, a switch in chromatin structure in the region between Ϫ2.7 and Ϫ2.4 kb is observed. The DHS at the Ϫ2.4-kb element disappears, until it is lost upon terminal differentiation and a new DHS at the Ϫ2.7-kb enhancer element is formed. Along with chromatin reorganization, transcription of the gene is up-regulated from a very low level in myeloblastlike cells to an almost 100-fold higher level in activated macrophage-like cells. These experiments correlate a high transcriptional level of the lysozyme gene with conditions where all enhancers are active and where the silencer element has been inactivated.
We showed that the chromatin structure displayed by the lysozyme locus in the various chicken cell types is faithfully reformed in lysozyme expressing and non-expressing cells of transgenic mice. This holds true for the DHSϪ and the nucleosomal phasing pattern as well as for the reorganization of the Ϫ2.4/Ϫ2.7-kb region after terminal macrophage differentiation (23,24), demonstrating that the same chromatin rearrangements take place in both species. Our experiments in transgenic mice also showed that for position-independent expression of the lysozyme locus in mature macrophages, the presence of all cis-regulatory elements is necessary (25,26). However, experiments with deletion mutants of the lysozyme locus in transgenic mice demonstrated that each enhancer region, despite their unique temporal regulation, is capable of driving macrophage-specific expression in mature macrophages (26). The question now remained of how the different cis-regulatory elements cooperate during earlier stages of cell differentiation. To this end, we have analyzed the time course of transcriptional activation of wild type and mutant lysozyme locus constructs during in vitro differentiation of myeloid precursor cells isolated from the bone marrow of transgenic mice. We show that constructs carrying a deletion of the late enhancer region including the silencer element are transcriptionally activated at the same developmental stage as the wild type locus carrying all cis-regulatory elements. The time course of activation of both constructs is indistinguishable, indicating a coincidence of structural reorganization of the early enhancers and the onset of mRNA synthesis. A construct in which the early enhancer has been deleted shows a delay in transcriptional activation. Our results suggest that the presence of the Ϫ2.4-kb silencer element on the wild type locus has no influence of the developmental onset of lysozyme locus activation, which is solely determined by the activity of the early enhancers.

EXPERIMENTAL PROCEDURES
Immunofluorescence Labeling and Cell Sorting-For each experiment four mice were killed by cervical dislocation. Bone marrow cells were flushed from femurs and tibias with phosphate-buffered saline (PBS). Cells were collected, washed once in PBS, and maintained on ice throughout the staining procedure. The cell suspension was depleted of red blood cells by hypotonic lysis with an ammonium chloride potassium buffer (Red Cell Lysis Buffer, Sigma). To remove debris, the suspension was centrifuged through a cushion of fetal calf serum (FCS; Life Technologies, Inc.). The remaining cells were washed once and resuspended in PBS supplemented with 3% FCS. After the suspension was passed through a cell strainer (Falcon/Becton Dickinson), 3.5 ϫ 10 7 cells in a volume of 200 l were used for staining. To suppress nonspecific binding, cells were first incubated for 5 min with 15 l of rat serum. Afterwards cells were incubated for 15 min with 16 l of ER-MP20-FITC (BMA), washed with PBS, 3% FCS and subsequently incubated (5 min) with 16 l of biotinylated ER-MP12 (BMA), followed by an addition of 16 l of streptavidin-Red670 and another incubation of 15 min. After two washes, the cells were resuspended in PBS, 3% FCS to a final concentration of 2 ϫ 10 6 cells/ml. Stained cells were analyzed and sorted on a FACStar plus (Becton Dickinson). The fluorescence intensity of individual cells was measured as relative fluorescence units. The purity of the sorted cell populations was determined by flow cytometry and exceeded 95%. In order to characterize cells differentiated in vitro by antibody staining, cells were stained with ER-MP12 and ER-MP20 as described above. In addition, cells were stained with R-phycoerythrin-coupled mAbs (Pharmingen) against B220 (RA3-6B2), Mac-1 (M1/70), F4/80 and IgM (isotype control). Stained cells were analyzed by flow cytometry.

IL-3-and M-CSF-stimulated Culture of Sorted Cell Populations-
The medium used for the culture of sorted cells was IMDM (Life Technologies, Inc.) supplemented with 10% FCS, 5% conditioned medium containing IL-3, 10% L-cell conditioned medium containing M-CSF, 2 mM glutamine, 100 units/ml penicillin, 100 units/ml streptomycin (Life Technologies, Inc.), and 1.5 ϫ 10 Ϫ4 M monothioglycerol. Conditioned media were prepared as described (27). After sorting the cell populations ER-MP12 hi /ER-MP20 Ϫ and ER-MP12 Ϫ /ER-MP20 hi were distrib-uted into 24-well plates (Greiner) depending on the number of kinetic time points and were cultured in a humidified environment with 5% CO 2 in air at a temperature of 37°C. Depending on the sorting efficiency 1.5-3 ϫ 10 4 ER-MP12 hi /20 Ϫ and 8 -10 ϫ 10 4 ER-MP12/20 hi cells were plated per well. When harvesting the cells in intervals of either 12 or 24 h over a period of 6 days, the adherent cell fractions were separated from the non-adherent fractions. When indicated, cells were stimulated with 5 g/ml LPS for 12 h. In order to obtain cells for antibody staining, cells were plated onto hydrophobic Petriperm cell culture dishes (Heraeus). Test experiments indicated that no difference in gene expression was found as compared with normal tissue culture dishes (data not shown).
RNA Isolation and cDNA Preparation-Total RNA was isolated from the adherent and non-adherent cells using 0.5 ml of RNAzol TM B (Biotecx Laboratories, Inc.) according to the manufacturer's instructions. cDNA of isolated total RNAs from the different samples was prepared using random hexamers as primers and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) in a reaction volume of 20 l under conditions recommended by the manufacturer. Per reaction 2 units of ribonuclease inhibitor (Life Technologies, Inc.) was added. cDNA was subsequently heated to 70°C for 5 min to inactivate reverse transcriptase.
Semiquantitative RT-PCR Analysis and Determination of Optimal cDNA Concentrations-To ensure the use of comparable amounts of RNA and cDNA for the analysis of the different samples, the relative expression level of the housekeeping gene HPRT was used as a standard for calibration. Only in the linear range of PCR reactions is it possible to quantitate the intensity of PCR signals. The linear range of PCR reactions is defined as the range in which the use of double cDNA amounts correlates with a duplication of the signal intensity. To define the linear range, serial 1:2 dilutions of the different cDNA samples were used to perform a PCR with primers specific for HPRT. For further analysis of the expression level of the endogenous mouse lysozyme and the different constructs of the transgenic chicken lysozyme, only those cDNA concentrations were used which gave rise both to signal intensities for HPRT within the linear range as well as comparable intensities among the different samples. The corresponding sample used for HPRT calibration was further diluted 1:2, 1:10, and 1:50 and used to perform a PCR with primers specific for mouse lysozyme. Due to the different expression levels of chicken lysozyme transgenes in the different mouse lines and in the various differentiation stages, it was necessary to carry out PCR reactions with a different range of dilutions for each mouse line. Except for mouse line dXK.2, 1 l of the following serial dilutions from the determined dilution of every sample which gave rise to a linear HPRT-PCR signal were used to analyze the different mouse lines: mouse line XS.0b: 1:2, 1:10, 1:50; mouse line XSdSS.28: 1:2, 1:4, 1:8. For mouse line dXK.2, 4, 2, and 1 l were used. Primers used were as follows: HPRT, 5Ј-CACAGGACTAGAACACCTGC-3Ј; 5Ј-GCTGGTGA-AAAGGACCTCT-3Ј; mouse lysozyme (m-lys), 5Ј-ACCCAGCCTCCAGT-CACCAT-3Ј, 5Ј-CAGTGCTTTGGTCTCCACGG-3Ј; chicken lysozyme (clys), 5Ј-GATCGTCAGCGATGGAAACGGC-3Ј, 5Ј-CTCACAGCCGGCA-GCCTCTGAT-3Ј. HPRT-PCRs were performed with 1.25 mM MgCl 2 and 18 pmol of each HPRT primer. PCRs for mouse lysozyme used 1.25 mM MgCl 2 and 15 pmol of each m-lys primer, whereas chicken lysozyme PCRs were performed with 1.0 mM MgCl 2 and 15 pmol of each c-lys primer. To every PCR reaction 1 l of the corresponding cDNA dilution was added. PCR reactions were done in a total volume of 30 l and with the use of 1.5 units of Taq polymerase (Life Technologies, Inc.). PCRs were carried out in a Trio-Thermoblock (Biometra) using a regimen of 94°C for 40 s, 55°C (HPRT) or 62°C (m-lys and c-lys) for 40 s, and 72°C for 1 min. After 35 cycles, the PCR was finished with a terminal elongation step of 72°C for 10 min. 15 l of each sample was loaded on a 6% polyacrylamide gel electrophoresis. Gels were stained with ethidium bromide and photographed with a Cybertech-Videoprinter (Mitsubishi) under 245 nm of UV light. Bands on digitized videoprints were quantified with the Image 1.35 program on a Macintosh computer. Signal intensities for mouse and chicken lysozyme were normalized to the HPRT signal, and the absolute normalized expression level was calculated from these values by multiplication with the dilution factors.

RESULTS
Experimental Strategy-The different cis-regulatory elements of the lysozyme gene are structurally activated at different macrophage differentiation stages as indicated by a changing pattern of DHS in the 5Ј-regulatory region of the lysozyme locus in various myeloid chicken cell lines (20) (Fig.  1A). The highest transcriptional level is observed in cell lines representing activated macrophages that display DNase I hypersensitivity at the position of all enhancer elements and where the DHS at the silencer element at Ϫ2.4 kb has completely disappeared. In order to evaluate the contribution of different cis-regulatory region to the developmental control of lysozyme expression, we analyzed the time course of transcriptional activation in the three different mouse lines indicated in Fig. 1, B and C (26). Mouse line XS.0b carries a construct with the full set of cis-regulatory elements, whereas the transgenes in mouse lines dXK.2 and XSdSS.28 carry either a deletion of the early enhancer region around Ϫ6.1 kb or a deletion of the late Ϫ2.7/Ϫ2.4-kb enhancer region, respectively. The presence or absence of domain bordering sequences has no influence on the expression of the different constructs in macrophages, as shown earlier (26).
In order to follow transcriptional activation of wild type and mutant lysozyme loci, we set up an in vitro differentiation system based on enriched myeloid precursor cells as outlined in Fig. 2. It has been shown that developing macrophage precursors can be characterized by a differential expression of the ER-MP12 and the ER-MP20 surface antigens (28,29). The most immature macrophage colony-stimulating factor (M-CSF) responsive precursors express a high level of the ER-MP12 and no ER-MP20 antigen (Fig. 2). During macrophage maturation, ER-MP12 expression is gradually switched off, whereas expression of ER-MP20 is switched on. Bone marrow monocytes express a high level of the ER-MP20 antigen and have lost the ER-MP12 antigen (30). We isolated ER-MP12 hi /20 Ϫ cells (ER-MP12 hi population) by fluorescence activated cell sorting in order to follow gene expression during early macrophage differentiation. To compare the mRNA levels in monocytes differentiated in vitro with the level found in bone marrow monocytes, we also analyzed ER-MP12 Ϫ /20 hi cells (ER-MP20 hi population). The isolated cell populations were cultivated under macrophage growth promoting conditions in the presence of M-CSF and interleukin-3 (IL-3) for a period of 6 days. Bacterial lipopolysaccharide (LPS) is known to promote macrophage ac-tivation and has been shown to stimulate expression of the chicken lysozyme gene in chicken and mouse macrophages (10,20,27,31). To examine at which stage of differentiation the cells acquire LPS responsiveness, cultures were treated with LPS for 12 h, either immediately after isolation or after 5 days of culture. Along with progressive macrophage maturation the cells acquire an adherent phenotype. Since we were interested in studying gene activation starting with a precursor population as immature as possible, we separated the cells into more mature adherent and immature non-adherent fractions. Cells were harvested at 12-or 24-h intervals as indicated in Fig. 2, and total RNA was prepared which was subjected to RT-PCR analysis for the expression of the following genes: HPRT as an internal calibration standard, the endogenous macrophagespecific mouse lysozyme M gene as a control marker for a successful and reproducible in vitro differentiation, and the chicken lysozyme transgene. In order to define the developmental stage at which transgene activation takes place, we immunostained cells at various time points of differentiation with labeled antibodies directed against markers indicative of different macrophage maturation stages (32).

Characterization of in Vitro Differentiated Myeloid Cells-
Macrophages developing from ER-MP12 hi populations in vitro have up to now not been characterized in detail. The ER-MP12 hi population consists mainly of small blast-like cells as described previously (30, data not shown). From day 3 of culture onwards, cells progressively acquire an adherent phenotype (see also Fig. 3). By day 6 most cells are adherent and from morphology and surface marker expression (Fig. 5) resemble monocytes. The ER-MP20 hi population resembles monocytic cells, which adhere quickly within 12 h of culture (see Fig. 3). During progressive differentiation cells of both populations acquire the characteristic features of macrophages, like irregular shapes and granules.
In order to further characterize cells developing in our culture system, we analyzed their proliferative capacity. In each respective differentiation culture we started with the same number of cells; hence, the comparison of the total amount of HPRT mRNA present in our RNA preparations from the different time points allowed an accurate description of relative cell numbers. In the same way, we could measure the proportion of adherent versus non-adherent cells. The result of this experiment is depicted in Fig. 3, which shows the relative increase in cell numbers and the changing proportion of adherent versus non-adherent cells in the different cell populations. An initial lag phase is observed for the ER-MP12 hi culture which is due to the presence of contaminating non-myeloid cells dying in the first 2 days of in vitro differentiation (30). Thereafter, both cell populations show the same proliferative capacity. The proportion of adherent cells in the ER-MP20 hi population is an order of magnitude higher compared with the ER-MP12 hi population. Mature ER-MP20 hi cells cease to proliferate toward the end of the culturing period. In contrast, the ER-MP12 hi population contains a significant number of proliferating non-adherent precursor cells which successively mature and adhere. This indicates that the non-adherent fraction of the ER-MP12 hi cell population contains the desired precursor cell types to use as a starting population for the analysis of the time course of transcriptional activation of different transgene constructs.
Transcriptional Activation of the Wild Type Chicken Lysozyme Locus during in Vitro Differentiation-Construct XS carries the full set of cis-regulatory elements of the lysozyme locus and is expressed at a high level in macrophages of trans-genic mice and independent of the chromosomal position of the transgene (26). Macrophage precursor cells from the bone marrow of transgenic mouse line XS.0b were isolated as described above and subjected to in vitro differentiation, and RNA prepared from the various cell populations was analyzed by RT-PCR. Since the ER-MP20 hi cell population is composed almost entirely of adherent monocytes, only the adherent cell fraction was examined. Fig. 4 shows the results of this experiment. One example for such an RT-PCR analysis is depicted in Fig. 4A. The time course of transcriptional activation of transgene and endogenous lysozyme gene was determined by quantifying PCR signals derived from the linear part of the amplification reaction (Fig. 4B) for each time point.
Expression analysis for chicken lysozyme and mouse lysozyme expression in the ER-MP12 hi fraction at day 0 revealed a very low level of expression of both genes. Expression of the chicken lysozyme transgene in the differentiating non-adherent fraction linearly increases from day 0.5 of in vitro differentiation onwards, whereas expression of the endogenous mouse lysozyme gene increases exponentially. The adherent ER-MP12 hi population shows a different time course of expression. Initially, both transgene and mouse lysozyme mRNA levels increase during differentiation culture. Surprisingly, progressive differentiation in culture leads to a decrease of transgene but not of mouse lysozyme mRNA levels. A similar phenomenon is observed with the differentiated ER-MP20 hi cell population, where transgene expression is initially high (probably as a result of activation via Fc receptors) and sharply declines after a couple of days in culture. However, in both populations LPS treatment leads to a reactivation of transgene expression. For comparison, we measured the chicken lysozyme expression level in thioglycolate elicited, relatively mature peritoneal macrophages of the same mouse line. The mRNA level in these cells that had been cultured for 1 day was comparable to the highest level found in in vitro differentiated cells but did not decrease during prolonged cell culture (data not shown). Only the more mature ER-MP20 hi population but not the non-adherent or the adherent ER-MP12 hi cell population responds to LPS treatment at early time points of differentiation. Expression of the endogenous mouse lysozyme gene was not induced by LPS treatment, as shown before (27,31). ysis of in Vitro Differentiating Cells-In order to determine the differentiation stage in which the chicken lysozyme transgene is activated in mouse cells, we analyzed the surface marker expression of differentiating cells. We stained cells at various time points of in vitro differentiation with antibodies directed against the ER-MP12 and ER-MP20 antigens, as well as against CD11b (Mac-1) (33) and F4/80 (34), the latter being surface markers characteristic for mature macrophages. A variant of the common leukocyte-specific antigen CD45R is expressed predominantly in B-cells and a subset of macrophages (35) and is recognized by the B220 antibody. An antibody against mouse IgM served as isotype-matched control (36). The results of these analyses are shown in Fig. 5. As predicted, in vitro differentiation of the ER-MP12 hi population leads to a successive down-regulation of the expression of the ER-MP12 antigen and to an up-regulation of the ER-MP20 antigen (30). The cells reach the double positive stage around days 2-3 of differentiation, about 8% of all cells display this phenotype at day 3 (data not shown). Subsequently, the cells express more and more epitopes characteristic for mature macrophages. Initially the expression of the B220 antigen is high, indicative of the presence of B-cell precursors in this population (37). Stimulated development of macrophages leads to a decrease in the number of B220 positive cells. The ER-MP20 hi population is characterized by a high level of expression of antigens specific for mature macrophages. Taken together, our analysis shows that chicken and mouse lysozyme gene expression is up-regu-lated from day 0.5 of differentiation onwards, where the cells have not yet reached the ER-MP12/ER-MP20 double positive stage and express only a low level of mature macrophage surface markers.

Determination of the Developmental Time Point of Chicken Lysozyme Gene Activation by Surface Marker Expression Anal-
The Early Enhancer Region Is Responsible for the Activation of the Lysozyme Locus at Early Stages of Development-We analyzed the time course of transcriptional activation of two different deletion mutants of the lysozyme locus during in vitro differentiation (26). Precursor cells of mouse lines XSdSS.28 and dXK.2 (Fig. 1) were isolated, and RNA from in vitro differentiated cells was analyzed by RT-PCR as described for mouse line XS.0b. Fig. 6, A and B, displays the results obtained for mouse line XSdSS.28, which carries a construct lacking the late enhancer region. The kinetics of transcriptional activation of this construct is undistinguishable from the construct carrying all cis-regulatory elements (Fig. 4). Here also we observe a continuous increase of expression in immature precursors followed by a decrease in more mature cell populations. The kinetics of transcriptional activation of construct dXK which lacks the Ϫ6.1-kb enhancer region was different (Fig. 6, C and  D). Expression in freshly isolated ER-MP12 hi cells at day 0 was high, probably as a result of contaminating more mature cells, which were lost due to their adherence after 1 day in the differentiation medium. Note that these contaminating cells also have already acquired LPS responsiveness (Fig. 6C, ER-MP12 hi a). Expression in precursor cells did not increase until day 2.5 of differentiation and thereafter was up-regulated with a kinetics similar to that observed for the other constructs. As with the other constructs, expression in the ER-MP20 hi cell population was high in the beginning but decreased toward terminal differentiation of the cells (Fig. 6, C and D, ER-MP20 hi a). Expression in the adherent ER-MP12 hi population was barely up-regulated. Expression from the XSdSSϪ and most likely also from the dXK construct was refractory to LPS stimulation at early differentiation stages but could be induced at later stages as it has been shown before (31). This holds true for all cell populations analyzed. The time course of activation of expression of the endogenous mouse lysozyme M gene in all in vitro differentiation cultures was basically indistinguishable, indicating that the time course of transgene activation is a function of the various constructs analyzed and is not due to differences in the differentiation kinetics of the cells.

The Wild Type Chicken Lysozyme Locus Is Transcriptionally Activated in Mouse Macrophages at the Same Developmental
Stage as in Chicken Macrophages-In our in vitro differentiation experiments we analyzed two populations of macrophage precursor cells representing two different maturation stages. Previous studies have shown that the ER-MP12 hi /20 Ϫ population contains about 50% morphologically undifferentiated blast cells, and the other half represents identifiable blasts cells of the myeloid, the lymphoid, and the erythroid lineage (30,38). 12% of the total cell population represents a mixture of granulocyte macrophage-colony forming cells (GM-CFCs) and macrophage-colony forming cells (M-CFCs/monoblasts) with a high proliferative potential (30). 3 The heterogeneous composition of this population explains the apparent lag phase in proliferation, since precursor cells of other hematopoietic lineages are unable to proliferate under our culture conditions. However, since we observe no reduction of relative cell numbers, we assume that myeloid precursors start proliferating immediately after plating. The ER-MP12 Ϫ /20 hi population contains predominantly monocytic cells (74%) with a few immature blasts cells (Ref. 30 and this study), which both immediately start to proliferate. Our experiments clearly demonstrate that under our culture conditions both sorted cell populations proliferate and simultaneously differentiate. The ER-MP12 hi /20 Ϫ population reaches the differentiation state of the ER-MP12 Ϫ / 20 hi population around day 6 as judged from mouse lysozyme and surface marker expression levels as well as from the ratio of adherent to non-adherent cells.
Our three-color surface marker analyses of differentiating ER-MP12 hi /20Ϫ cells indicate that they transiently mature 3 M. de Bruijn and P. Leenen, personal communication.
A). All samples were stained with biotinylated (ϩRED 670) ER-MP12, ER-MP20-FITC and the corresponding R-phycoerythrin-labeled third mAb specific for either of the indicated surface markers. A mAb against mouse IgM was used as an isotype-matched control (IgG 2b) for the macrophage-specific mAbs Mac-1 and F4/80. For analysis of the third mAb at day 0 live gates were set on either ER-MP12 hi /20 Ϫ (A) or ER-MP20 hi /12 Ϫ (B). Histograms in A were generated from 100,000 (bone marrow), about 1000 (day 0), 500 (day 1), 1000 (day 3), and 2000 (day 6) events; histograms in B were generated from about 5000 (day 0) and 500 (day 6) events. into cells expressing both the ER-MP12 and the ER-MP20 antigen around days 2-3 of in vitro differentiation. A cell population isolated from the bone marrow expressing this surface marker combination has been shown to consist of some M-CFCs and promonocytes (30). The increase in chicken lysozyme expression begins at day 0.5 of differentiation whereby halfmaximal expression is reached by day 3. The onset of transgene expression occurs therefore most likely at the GM-CFC stage (which is analogous to the myeloblast stage in the chicken system), indicating a concordance in developmental regulation between the two species (20). Since also the same developmentally controlled chromatin rearrangements occur in both species, we were able to draw relevant conclusions from the analysis of wild type and mutant gene loci in transgenic mice.
Transgene expression decreased at later macrophage differentiation stages. This phenomenon was observed for all constructs tested. However, it was not observed with thioglycolateelicited peritoneal macrophages kept in culture for up to 2 weeks, indicating that the lysozyme transgene is expressed at a high level in in vivo differentiated cells. The uniformly high expression level of the endogenous mouse lysozyme gene as well as the reactivation of a high level of transgene expression by LPS treatment argues against a general decrease of cell viability in our culture system. It rather indicates that the cells reach a quiescent differentiation stage which does not support transgene expression without a further signal. In vivo, monocytes migrate into various tissues and differentiate into various mature macrophage types. In most cases such cells also cease expression of the endogenous mouse lysozyme gene. We could show that expression of the chicken lysozyme transgene, in contrast to the mouse lysozyme gene, is a faithful marker for macrophage activation (27,31). Our experiments indicate that transgene expression is up-regulated until the cells reach the monocyte stage where they normally would leave the bone marrow is then down-regulated and is only reactivated after further macrophage differentiation. Thioglycolate-elicited macrophages obviously have received an additional differentiation signal required to maintain a high transgene expression level, which in addition can be further enhanced by LPS treatment (31).
The Role of the Different Cis-regulatory Elements in Lysozyme Gene Regulation-The various enhancer elements on the lysozyme locus assemble transcription factors and thus become DNase I-hypersensitive at different stages of macrophage development. Our studies of the time course of activation, as well as earlier chromatin structure analyses, point to a different role of various cis-regulatory elements with respect to the coordinated activation of the lysozyme locus during development. In early macrophage precursor cells the early enhancers, the promoter, and the silencer element are active, whereas the late Ϫ2.7-kb enhancer is inactive (20,22,23). Hence, several scenarios could be envisaged. Either the early (Ϫ6.1-kb and Ϫ3.9-kb) enhancers and the promoter drive lysozyme transcription at a low frequency irrespective of the presence of the silencer at Ϫ2.4 kb or the functional silencer element competes with the enhancers and inhibits transcription at early differentiation stages. This would allow the gene to be transcribed only after the developmentally controlled inactivation of the silencer element and the simultaneous activation of the late Ϫ2.7-kb enhancer. The third possibility would be a developmentally controlled reorganization of the early enhancers by transcription factors synthesized later in differentiation which in turn would be necessary for their interaction with the basal transcription machinery. The two latter models would imply that the structural reorganization of the early enhancers is uncoupled from their ability to drive transcription.
Each of these scenarios could be experimentally distinguished. If the two latter models were true, mutants of the lysozyme locus carrying deletions of early or late enhancer regions should have shown characteristic differences in the time course of developmental activation as compared with a locus carrying all cis-regulatory elements. Such differences should also have been observed if the presence of the Ϫ2.4-kb silencer element had any influence on the onset of lysozyme expression at early differentiation stages, i.e. if it would repress expression of the lysozyme locus in early precursors. However, the time course of transcriptional activation of the chicken lysozyme transgene, with or without the silencer element, is identical. Both transgenes up-regulate transcription at day 0.5 of in vitro differentiation and decrease expression at later differentiation stages with similar kinetics. This is in contrast to the construct carrying a deletion of the Ϫ6.1-kb enhancer region. Here, in concordance to the chromatin studies, we observe a 2-day delay in the onset of transcriptional activation. At day 2.5 to 3 the cells reach the ER-MP12/ER-MP20 double positive (promonocytic) stage. At this stage, in analogy to the chicken system, the Ϫ2.7-kb enhancer is reorganized and thus activated. Our results also indicate that the presence of the early Ϫ3.9-kb enhancer alone is not sufficient to activate transcription in early progenitors.
Our results demonstrate that the absence of the silencer element does not alter the time course of transcriptional upregulation, implying that this element does not repress the action of the early enhancers. Although we have no evidence to suggest this from the present analysis, we prefer a model in which the Ϫ2.4-kb element is involved in repressing the macrophage-specific Ϫ2.7-kb enhancer element at early developmental stages of myeloid differentiation. The same element might also be responsible for the repression of the Ϫ2.7-kb enhancer in the chicken oviduct, where the gene is expressed under steroid hormone control. Support for this idea comes from experiments that demonstrate the presence of a DHS at the Ϫ2.4-kb element in the chicken oviduct, where the Ϫ2.7-kb enhancer is not hypersensitive and thus not active (18). Previous chromatin analyses have demonstrated that the silencer element and the immediately juxtaposed enhancer element are each organized in a positioned nucleosome and may form an integrated cis-regulatory element. The spacing of binding sites is such that they may face the same side on each nucleosome, thus bringing them in close contact (23). Chromatin rearrangements at the Ϫ2.4-kb and at the Ϫ2.7-kb elements are strictly parallel, and the appearance of MNase and DNase I-hypersensitive sites at the enhancer correlates with the disappearance of such sites at the negative regulatory element, indicating that factor binding at both elements is mutually exclusive.
Taken together our data indicate that initial locus activation is performed by the interaction of the early enhancers with the promoter. Maximal transcriptional activity, which is necessary during a bacterial attack (simulated by LPS treatment), is achieved by the inactivation of the negative regulatory element and the simultaneous activation of the Ϫ2.7-kb enhancer. In addition, the activity of all enhancers as well as the promoter can be modulated by LPS and other macrophage activating agents (10; 31; 39), 4 indicating that the expression status of the chicken lysozyme locus, as an endogenous gene in chicken and as a transgene in the mouse, is strongly dependent on the physiological status of a macrophage cell.


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Structure and Folding of Nascent Polypeptide Chains during Protein Translocation in the Endoplasmic Reticulum*

To investigate the role of protein folding and chaperone-nascent chain interactions in translocation across the endoplasmic reticulum membrane, the translocation of wild type and mutant forms of preprolactin were studied in vivo andin vitro. The preprolactin mutant studied contains an 18-amino acid substitution at the amino terminus of the mature protein, eliminating a disulfide-bonded loop domain. In COS-7 cells, mutant prolactin accumulated in the endoplasmic reticulum as stable protein-protein and disulfide-bonded aggregates, whereas wild type prolactin was efficiently secreted. In vitro, wild type and mutant preprolactin translocated with equal efficiency although both translation products were recovered as heterogeneous aggregates. Studies with translocation intermediates indicated that aggregation occurred co-translationally. To evaluate the contribution of lumenal chaperones to translocation and folding, in vitro studies were performed with native and reconstituted, chaperone-deficient membranes. The absence of lumenal chaperones was associated with a decrease in translocation efficiency and pronounced aggregation of the translation products. These studies suggest that chaperone-nascent chain interactions significantly enhance translocation and indicate that in the absence of such interactions, aggregation can serve as the predominant in vitro protein folding end point. The ramifications of these observations on investigations into the mechanism of translocation are discussed.

To investigate the role of protein folding and chaperone-nascent chain interactions in translocation across the endoplasmic reticulum membrane, the translocation of wild type and mutant forms of preprolactin were studied in vivo and in vitro. The preprolactin mutant studied contains an 18-amino acid substitution at the amino terminus of the mature protein, eliminating a disulfide-bonded loop domain. In COS-7 cells, mutant prolactin accumulated in the endoplasmic reticulum as stable protein-protein and disulfide-bonded aggregates, whereas wild type prolactin was efficiently secreted. In vitro, wild type and mutant preprolactin translocated with equal efficiency although both translation products were recovered as heterogeneous aggregates. Studies with translocation intermediates indicated that aggregation occurred co-translationally. To evaluate the contribution of lumenal chaperones to translocation and folding, in vitro studies were performed with native and reconstituted, chaperone-deficient membranes. The absence of lumenal chaperones was associated with a decrease in translocation efficiency and pronounced aggregation of the translation products. These studies suggest that chaperone-nascent chain interactions significantly enhance translocation and indicate that in the absence of such interactions, aggregation can serve as the predominant in vitro protein folding end point. The ramifications of these observations on investigations into the mechanism of translocation are discussed.
Current models of protein translocation across the mammalian endoplasmic reticulum (ER) 1 depict translocation as a process in which vectorial transport accompanies formation of a tight junctional complex between the ribosome and the protein conducting channel with the free energy for translocation provided by passive diffusion (1,2). In alternative models, vectorial transport may also be driven through interaction of the nascent chain with lumenal molecular chaperones (3)(4)(5)(6), as well as structural modifications of the nascent chain, i.e. protein folding, disulfide bond formation and, in many cases, ad-dition of N-linked oligosaccharides, that occur coincident with translocation (7)(8)(9)(10)(11). In the latter model, interactions between the nascent chain and lumenal molecular chaperones are thought to prevent retrograde transport through the translocation pore and thus bias movement of the nascent chain into the lumenal compartment (3)(4)(5)(6)12).
Recent reconstitution experiments have identified the minimum subset of ER proteins necessary for in vitro protein translocation in the mammalian ER (13,14). In the minimal system, the translocation machinery is comprised of the signal recognition particle receptor which functions in the targeting of ribosome/nascent chain complexes to the ER, the Sec61p complex, which is thought to serve as a ribosome receptor and translocation channel, and, in some instances, the integral membrane protein TRAM, which participates in signal sequence recognition (13)(14)(15). The identification of the Sec61p complex as the primary ribosome receptor and translocation channel suggests that ribosome association with Sec61p could provide the aqueous pathway for nascent chain transit into the lumen (15,16).
It is clear from molecular genetic and biochemical studies that hsp70 proteins perform an essential function in protein translocation across the yeast ER and the mitochondrial inner membrane (17)(18)(19)(20)(21)(22). From these studies, it has been proposed that hsp70 proteins promote unidirectional transport by binding to the nascent chain as it emerges into the lumen (matrix). It has not yet been established whether the interaction of the hsp70 proteins with the nascent chain drives vectorial transport by a thermal ratchet mechanism (3)(4)(5), or, alternatively, by a conformationally driven, motor process (23,24). Furthermore, there is disagreement as to whether the role of hsp70 proteins is limited to translocation events that occur posttranslationally (20), or alternatively, whether hsp70 proteins are required for co-and post-translational translocation (25). Interestingly, it has been demonstrated that BiP is necessary for the complete translocation of prepro-␣-factor, a precursor known to translocate post-translationally (26). In the absence of BiP function, pro-␣-factor is unable to fully transit to the ER lumen (26). This defect, referred to as stalling, is quite similar to a translocation defect observed in mammalian microsomes that have been depleted of their lumenal contents (5). In mammalian microsomes, the loss of lumenal proteins causes a disruption of the translocation reaction at a point subsequent to signal sequence cleavage, and results in the accumulation of the signal-cleaved nascent chains that are unable to efficiently transit to the vesicle lumen (5).
The analysis of the energetics of protein translocation is made difficult by the fact that protein translation, translocation, and folding are coincident processes and furthermore, that protein folding occurs in an environment, the ER lumen, which is highly enriched in molecular chaperones and protein folding enzymes. To study the contribution of protein folding and lumenal chaperone-nascent chain interactions to translo-cation, the translocation behavior of wild type and mutant forms of preprolactin (pPL) were analyzed in vivo and in vitro. The mutant preprolactin used in this study contains an 18amino acid substitution at the NH 2 terminus of the mature protein, eliminating a small disulfide-bonded loop domain (27,28). Whereas wild type (WT) prolactin was efficiently secreted in vivo, the folding mutant (FA) accumulated in the ER as large protein-protein and disulfide-bonded aggregates. In vitro, both WT and FA forms of prolactin were translocated with similar efficiencies but were recovered as mixed aggregates. Furthermore, aggregation was apparent co-translationally. In the absence of lumenal chaperones, aggregate formation was markedly enhanced and was accompanied by a reduction in translocation efficiency. On the basis of these data, we propose that lumenal protein-nascent chain interactions are paramount to efficient translocation and in their absence, irreversible protein-protein aggregation may serve as the predominant protein folding end point.
Clones and Vectors-Clone pPC-BP1 was constructed by excision of a full-length bovine preprolactin cDNA from the plasmid pGEMBP1 (31) with HindIII and EcoRI, and subcloning of the HindIII/EcoRI fragment into HindIII/EcoRI-digested mammalian expression vector pCDNA3 (Invitrogen, La Jolla, CA).
Mutant clone pPL-FA was constructed by 5Ј add-on/recombinant PCR (32) using pGEMBP1 as template. In the mutant clone FA, the first 18 amino acids of mature prolactin are: YHCDGFQNEQIYTDL-EMN, whereas in wild type prolactin, the sequence is: TPVCPNG-PGNCQVSLNDL. In wild type prolactin, a disulfide bond is formed between amino acids 4 and 11 (28). In the mutant the entire disulfidebonded loop domain has been exchanged with a random sequence.
Products 1 and 2 were gel purified and used as template cDNA in a second PCR reaction with oligonucleotides product 1, sense, and product 2, antisense. PCR was performed as described above and the product, representing full-length pPL-FA cDNA, gel purified. Full-length pPL-FA cDNA was digested with HindIII/BamHI and subcloned into the vector pCDNA3 for transient transfection experiments. Positive clones, identified by antibiotic selection and restriction mapping, were subjected to dideoxy sequencing prior to transfection studies, to ensure the accuracy of the mutations.
Pulse-Chase/Immunoprecipitation-Pulse-chase studies were performed 40 -48 h post-transfection as described in Ref. 33. Monolayer cultures in 60-mm dishes were washed twice in methionine and cysteine-free Dulbecco's modified Eagle's medium and the respective cellular amino acid pools depleted by incubation in methionine and cysteine-free Dulbecco's modified Eagle's medium for 20 min at 37°C. The labeling reaction was subsequently performed by addition of 200 Ci/ml [ 35 S]methionine/cysteine (Pro-Mix; Amersham) in 300 l of methionine/ cysteine-free Dulbecco's modified Eagle's medium for 15 min at 37°C. Following the labeling period, isotope-supplemented media was removed, and cells washed in complete Dulbecco's modified Eagle's medium containing 2 mM methionine, 0.5 mM cysteine, 1% fetal calf serum (chase media). At the indicated time points, the chase media (0.5 ml) was removed and the cells harvested by scraping into ice-cold PBS. Cells were washed 2 times in PBS and lysed by addition of 1 ml of lysis buffer (25 mM Tris/Cl (pH 7.4), 300 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 25 g/ml soybean trypsin inhibitor, 5 mM EDTA). After a brief sonication, samples were supplemented with bovine serum albumin, to a final concentration of 2.5 mg/ml, and centrifuged to remove particulate material. Lysates were pre-cleared by addition of either 40 l of a 50% slurry of Protein A-Sepharose or the equivalent volume of Pansorbin (Calbiochem) and indirect immunoprecipitations performed by addition of rabbit antisheep prolactin (U. S. Biochemical Corp., Cleveland, OH) (1:250 dilution) and overnight incubation at 4°C. Immune complexes were collected by addition of 40 l of a 50% slurry of Protein A-Sepharose and incubation at room temperature for 30 min. Protein A-Sepharose resin was collected by centrifugation (2 min, 2,000 ϫ g), extensively washed with lysis buffer, and resuspended in PBS. Radiolabeled prolactin was eluted by addition of 40 l of 0.5 M Tris, 5% SDS, 0.1 M DTT and heating for 20 min at 65°C and 5 min at 95°C. Samples were resolved on 12.5% SDS-PAGE gels or Tris-Tricine gels as described previously (34). In experiments in which disulfide bond formation was studied, monolayers were washed in ice-cold PBS, and prior to lysis, free sulfhydryls alkylated by addition of 20 mM N-ethylmaleimide (NEM) for 20 min on ice, as described in Ref. 35.
Isolation and Protease Digestion of COS-7 Microsomal Fraction-Microsomal membranes from transfected COS-7 cells were prepared as follows: 40 h post-transfection, cells from two 100-mm culture dishes were pulse labeled as described above, the labeling media removed, and the cells recovered by scraping into ice-cold hypotonic lysis buffer (10 mM K-HEPES, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml pepstatin). The cells were washed 1 time in lysis buffer, resuspended to 1 ml, incubated on ice for 15 min, and homogenized (20 strokes) with a tight fitting Dounce homogenizer. The homogenate was adjusted to 250 mM sucrose and centrifuged for 10 min at 700 ϫ g (4°C). The supernatant from this step was removed and the centrifugation step repeated. The final supernatant was centrifuged for 15 min at 70,000 rpm in the Beckman TLA100.3 rotor. The resulting pellet fraction, representing the post-nuclear membranes, was resuspended in 250 mM sucrose, 10 mM K-HEPES (pH 7.2), 50 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , to a final volume of 0.5 ml. Protease digestions were performed on ice for 30 min with 50 g/ml proteinase K. Where indicated, Triton X-100 was present at 0.5%. Protease digestions were quenched by addition of phenylmethylsulfonyl fluoride to 3 mM and after a 15-min incubation on ice, the membranes were solubilized with lysis buffer, and radiolabeled prolactin translation products isolated by immunoprecipitation.
Analysis of Aggregation State-Pulse-chase studies were performed as described above and incubations quenched by addition of ice-cold PBS supplemented with 20 mM NEM. Cell lysates were prepared as described (35) and cleared of large aggregates by centrifugation for 15 min at 15,000 ϫ g (4°C). Lysates were overlaid onto 8 -35% sucrose gradients prepared in lysis buffer supplemented with 0.2% Triton X-100. Gradients were prepared and harvested with a Buchler Auto-Densi Flow apparatus (Buchler Instruments, Lexana, KY). Gradients were centrifuged for 16 h at 39,000 ϫ rpm in the Beckman SW-40 rotor (4°C). 850-l fractions were collected, and indirect prolactin immunoprecipitations performed as described above.
Protein Translation/Translocation-In vitro protein translation/ translocation experiments were performed as described in Ref. 36. All translations were performed using reticulocyte lysate, prepared as in Ref. 37, and canine pancreas rough microsomes, prepared as described in Ref. 38.
Membrane Reconstitution-Reconstitution of translocation competent membranes from detergent-solubilized canine pancreas rough microsomes was performed as described in Ref. 39.

Generation of Preprolactin Structural Mutant FA-
The preprolactins are members of a family of hormones that include the growth hormones and placental lactogens (27,28). These proteins share extensive amino acid sequence homology and are thought to display a conserved, general, three-dimensional structure (27,40). The mammalian prolactins can be readily distinguished from the growth hormones by a small, disulfidebonded loop (amino acid residues 4 -11) present at the amino terminus (27,28). The disulfide-bonded loop immediately precedes one of the four ␣-helical segments characteristic of this family of hormones and is diagramatically illustrated in Fig. 1. By PCR mutagenesis, the sequence encompassing this disulfide-bonded domain was exchanged with a random amino acid sequence, lacking the residues 4 -11 disulfide bond pair, to yield a mutant referred to as pPL-FA (Fig. 1).
It has been established that disulfide bond formation can occur immediately upon the appearance of a relevant cysteinecysteine pair in the ER lumen (41). In the prolactin mutant FA, the NH 2 -terminal domain lacks the cysteine pair present in the wild type protein, and therefore a protein folding event which likely occurs very early in translocation, perhaps immediately upon access of the nascent chain to the ER lumen, cannot occur. It was postulated that mutations within this discrete structural domain would significantly disrupt the protein folding pathway of prolactin, and thereby prove useful in investigations on the contributions of protein folding and lumenal protein interactions to protein translocation.
FA Is Neither Secreted Nor Degraded-Exit of secretory proteins from the ER occurs coincident with structural maturation and is the underlying basis for the variations in the rate of secretion observed between proteins in a given cell (42)(43)(44)(45). To assay for disruptions in the folding behavior of pPL-FA, pulsechase studies were performed in transfected COS-7 cells expressing either pPL-WT or pPL-FA. The results of these studies are depicted in Fig. 2. Under the described conditions, prolactin-WT is rapidly secreted with a half-time of 25 min (n ϭ 4) ( Fig. 2A, WT). Prolactin-FA, in contrast, remained predominately cell associated throughout the 90-min chase time ( Fig.  2A, FA), indicating that prolactin-FA displays a significant defect in protein folding.
Proteolytic degradation via a non-lysosomal protein degradation pathway is a common fate of transport-incompetent proteins (46,47). To assess the stability of the secretion incompetent prolactin-FA, extended pulse-chase studies were performed. In these experiments, it was observed that at chase periods of up to 8 h, the vast majority of the prolactin-FA was neither secreted nor degraded (Fig. 2B, compare WT versus FA). These results suggest that prolactin-FA is either not a substrate for the relevant proteases or, alternatively, has not gained access to the degradation compartment.
FA Is Retained in a Membrane Compartment-Recent studies on ER-associated protein degradation have provided evidence of a novel pathway which functions to transport malfolded proteins from the ER to the cytosol, for subsequent degradation by the proteasome complex (48 -50). To determine if prolactin-FA was retained within a membrane compartment, presumably the ER, nascent chains were pulse-labeled and a microsomal fraction prepared from the labeled cells. The protease accessibility of prolactin-FA and prolactin-WT in the microsomal fraction was then ascertained by digestion with exogenous proteases. The results of these experiments are shown in Fig. 3. Both WT and FA forms of prolactin were equivalently protected from digestion with exogenous proteases in the absence (lanes 2 and 5) but not the presence (lanes 3 and 6) of detergent. The absolute degree of protease protection varied from 40 to 70% between experiments although in all cases the relative degree of protease protection of prolactin-WT and prolactin-FA was nearly identical. 2 These data, in combination with those presented in Fig. 2, indicate that the described mutation in the NH 2 -terminal disulfide-bonded loop domain yields a form of prolactin that is efficiently translocated yet remains in a structural state that is unsuitable for trans-2 R. L. Haynes and C. V. Nicchitta, unpublished observations. port through the secretory pathway or retrograde transport to the cytosol.
FA Forms Protein-Protein and Disulfide-bonded Aggregates-It has been reported that nascent chains may form protein-protein, as well as disulfide-bonded aggregates, during protein folding in the ER (44,45,51). To assess the structural status of translocated WT and FA forms of prolactin, transfected cells were metabolically labeled, chilled, treated with 20 mM NEM to block artifactual disulfide bond formation (35), and detergent lysates prepared for analysis by velocity sedimentation (Fig. 4A). In cells expressing prolactin-FA, metabolic labeling was performed in the presence (FA ϩ DTT), or absence (FA Ϫ DTT) of 20 mM DTT. It has previously been demonstrated that in the presence of DTT, disulfide bond formation in the ER is blocked, in many cases leading to a reversible dis-ruption in protein folding (35,44). Following centrifugation and harvesting of the gradients, prolactin was recovered by immunoprecipitation and analyzed by SDS-PAGE. As shown in Fig.  4A, prolactin-WT was recovered at the top of the gradient, consistent with a monomeric status for the export competent protein. In contrast, prolactin-FA was present as heterogeneous aggregates, widely dispersed through the gradient fractions and displaying sedimentation coefficients of 10 -40 S (Fig.  4A; data not shown). The relative distribution of FA was unaffected when labeling was performed under reducing conditions, indicating that protein-protein interactions contribute substantially to aggregate formation. These results clearly indicate that the structural state of the mutant prolactin differs markedly from the wild type. In further studies, in which cells were treated with cross-linking reagents prior to lysis, the ER chaperone BiP was identified in the FA aggregate pool. 2 The use of cross-linking reagents was, however, necessary to identify such interactions.
As prolactin-FA lacks the cysteine pair necessary for formation of the disulfide-bonded NH 2 -terminal loop, we postulated that protein folding would necessarily be disrupted at the level of disulfide bond formation. To ascertain the state of disulfide bond formation, WT and FA transformants were subjected to a pulse-chase study, and the cell-associated prolactin recovered at 0, 30, and 60 min. Following immunoprecipitation, samples were run on reducing and non-reducing gels. Disulfide bond formation stabilizes protein 3°protein structure, resulting in faster migration of the oxidized proteins on SDS-PAGE (35). This phenomenon was clearly evident with respect to WTprolactin, in which oxidized prolactin was observed to migrate faster than reduced prolactin on SDS-PAGE gels (Fig. 4B, lanes  1-3 versus lanes 7-9). Prolactin-FA rapidly formed large disulfide-bonded aggregates and, under non-reducing conditions, was preferentially recovered in the stacking gel and stacking gel interface (Fig. 4B, lanes 4 -6 versus lanes 10 -12). In these experiments, large, disulfide-bonded prolactin-FA aggregates was observed at the zero time point (15-min labeling period), indicating that the formation of disulfide-bonded aggregates was quite rapid.
Translocation Behavior of pPL-WT and pPL-FA in Vitro-In vitro translocation systems have proven valuable in the identification and analysis of the molecular stages of translocation (5,31,36,52). Having defined the structural basis of the in vivo secretion defect observed with prolactin-FA, the translocation and folding behavior of prolactin-WT and prolactin-FA were investigated in vitro using both native rough microsomes and reconstituted vesicles lacking lumenal proteins. Depicted in Fig. 5, are the results of an in vitro translocation study of the WT and FA forms of pPL. In vitro, secretory protein translocation is commonly assessed by two criteria, signal sequence cleavage and insensitivity of the mature protein to digestion by exogenous proteases. By these criteria, pPL-FA behaves identically to pPL-WT. Thus, when translated in the presence of rough microsomes, both forms are subject to signal sequence cleavage (Fig. 5A, lanes 1, 2, and 4) and mature prolactin, but not the precursor, are protected from digestion with exogenous proteases (Fig. 5A, lanes 3 and 5). Clearly, both in vivo and in vitro, the protein folding defect associated with prolactin-FA was without effect on translocation.
To determine the structural state of the in vitro translocated WT-and FA-prolactin, completed translocation reactions were treated with NEM, solubilized, and the structural status of the nascent chains analyzed by velocity sedimentation. In contrast to the in vivo results (Fig. 4A), in which the WT-prolactin was recovered in a fully folded, monomeric state, both WT-and FA-prolactin formed large, heterogeneous aggregates in vitro  Fig. 2. Where indicated, pulse labeling was performed in the presence of DTT. Following metabolic labeling, cells were washed, chilled on ice, and treated with 20 mM NEM to block artifactual disulfide bond formation. Cell lysates were prepared, cleared of large aggregates by centrifugation for 15 min at 15,000 ϫ g, and overlaid onto 8 -35% sucrose gradients supplemented with 0.2% Triton X-100. Gradients were centrifuged for 16 h at 39,000 ϫ rpm in the Beckman SW-40 rotor (4°C). Fractions were collected, indirect prolactin immunoprecipitations performed, and the immunoprecipitates analyzed by SDS-PAGE and PhosphorImaging. Digital images of the gels are depicted. B, COS-7 cells were transfected with wild type and mutant forms of pPL. Forty hours post-transfection, cells were pulse labeled as described in the legend to Fig. 2, washed, and the chase period initiated. At the indicated time points, cells were lysed, and the radiolabeled prolactin recovered by indirect immunoprecipitation. Immunoprecipitates were split into two equivalent aliquots and solubilized for SDS-PAGE in either the presence or absence of 100 mM DTT. Samples were resolved by SDS-PAGE; a digital image derived from PhosphorImager analysis of the gel is depicted. The boundary between the separating and stacking gel is shown to the left. The location of mature prolactin is indicated by the asterisk. (Fig. 5B). Aggregate formation was more pronounced for prolactin-FA, with the majority of the protein sedimenting as large (Ͼ20 S) particles ( Fig. 5B; data not shown). A quantitative depiction of the data is shown in Fig. 5C. It is apparent from comparison of Figs. 4B and 5C that the folding defect seen for FA-prolactin in vivo can be recapitulated in vitro. It is also evident from this comparison that the efficiency of WT-prolactin folding is significantly and substantially reduced in the in vitro translation/translocation system.
Co-translational Aggregation of Translocation Intermediates-The observation that WT-prolactin undergoes substantial aggregation under the experimental conditions used for in vitro translation/translocation prompted immediate concern. Aggregation of incompletely folded nascent polypeptide chains can be a thermodynamically favorable process, and is a commonly observed phenomena in in vitro folding studies (51,53,54). For this reason, it was important to determine whether the aggregation process observed in the in vitro system occurs co-translationally or post-translationally, and thus whether aggregation could contribute a driving force to translocation. It is clear from recent studies that many precursor proteins form reversible protein-protein aggregates during early stages of protein folding in the ER (44,45,51), although to date, cotranslational aggregation of nascent chains has not been demonstrated.
To evaluate the structural status of translocation intermediates, a series of stable, truncated translocation intermediates was studied. In these experiments, nascent pPL chains of 86 and 169 amino acids were translated from truncated mRNA transcripts. Such transcripts, because they do not possess a termination codon, direct synthesis of the nascent chain, but remain in stable association with the ribosome (55,56). As depicted in Fig. 6, panels A and D, greater than 80% of membrane-associated 86-amino acid pPL precursor (pPL 86-mer), either in association with the ribosome (not shown) or upon puromycin-induced release into the ER lumen (ϩ Puro), was recovered at the top of the sucrose gradients, and thus, by these criteria, does not undergo extensive aggregation. pPL 86-mer, when bound to the ribosome, is not a substrate for the signal peptidase and does not extend into the ER lumen (31,36,52). pPL 169-mer, in contrast, is of sufficient length to undergo signal peptide cleavage while remaining in association with the ribosome, and thus has gained access to the ER lumen (34,57). By comparing the relative migration in sucrose gradients of full-length prolactin, synthesized in vivo (Fig. 4A), with the signal cleaved pPL 169-mer translocation intermediate (Fig. 6,  B-D), it is clear that at an early stage of translocation, in vitro translocation nascent chains can enter a heterogeneous aggregate pool. We have so far been unable to identify stable complexes of the translocation intermediates with lumenal chaperones, such as BiP or PDI (data not shown). It thus appears that aggregates are forming with partially folded precursors present in the ER lumen during translocation (see "Discussion"). When pL 169-mer intermediates are released from the ribosome prior to solubilization, aggregation was exacerbated, and the protein was recovered in fractions throughout the gradient (Fig. 6, C and D). As expected from previous results, the FA form of pL 169-mer behaved similarly, although displaying a higher propensity for aggregation (data not shown).
Structural State of Nascent Chains in Chaperone-depleted Membranes-The results presented in Figs. 5 and 6 make evident an important point. Although the in vitro translation/ translocation system can accurately reproduce the signal cleavage and translocation events that accompany translocation, the structural status of the translocated proteins, be they wild-type or mutant, are significantly different from that observed in vivo. In light of experimental evidence indicating that lumenal proteins support both vectorial translocation and efficient protein folding, the structural state of nascent chains synthesized in the presence of native RM or vesicles depleted of lumenal chaperones was compared. Given the propensity toward aggregation seen in native membranes, we postulated that in the absence of chaperones, aggregation would be exacerbated, and thus might contribute the predominant driving force for translocation. Previously we reported that reconstitution of translocation competent vesicles from detergent-solubilized ER membranes results in the near complete loss of the lumenal contents and a marked reduction in the efficiency with which signalcleaved precursors are fully transported (39,58). Similar findings are illustrated in panels A and B of Fig. 7. In comparing native and reconstituted RM, both vesicle populations were observed to efficiently mediate translocation to the point of signal sequence cleavage (Fig. 7A, compare lanes 1, 2, and 5). Protease protection studies, a measure of net translocation, demonstrate, however, that the rRM do not translocate the signal-cleaved precursors as efficiently as RM (Fig. 7A, lanes 2  and 3 versus 5 and 6). This observation is further substantiated in the sedimentation studies detailed in Fig. 7B. In the experiment illustrated in Fig. 7B, membrane fractions were isolated from the completed translation reactions by centrifugation, and the pellet and supernatant samples analyzed for the recovery of associated translation products. In the absence of membranes (Fig. 7B, lanes 1, 2, 5, and 6), the nascent precursor (p) is recovered in the supernatant fraction, whereas in the presence of native membranes, the signal cleaved, or mature form (m) co-sediments with membrane fraction (lanes 3 and 4). In contrast, when translations are performed in the presence of reconstituted membranes, substantial quantities of the signalcleaved, mature protein are recovered in the supernatant fraction (Fig. 7B, lanes 7 and 8). The recovery of the signalcleaved form of prolactin in the supernatant fraction indicates that the precursor had undergone targeting and translocation to the point of signal sequence cleavage. A substantial fraction of the signal-cleaved precursor, however, transited free from the translocon to the cytoplasm and was thus recovered in the supernatant. The structural state of the prolactin synthesized in the presence of control and reconstituted membranes was further analyzed be velocity sedimentation (Fig. 7C and D).
Consistent with previous results (Fig. 5), pPL-WT, when translated in the presence of RM, is recovered throughout the gradient, with the predominant fraction present at the top of the gradients. When translated in the presence of reconstituted RM, WT-prolactin was preferentially recovered in the aggregate pool, with a distribution markedly similar to that observed for the prolactin-FA folding mutant in vivo (Fig. 4) and in vitro (Fig. 5). In point, when translated in the presence of reconstituted membranes, the translocation behavior and structural state of the WT-and FA-prolactin were very similar, indicating that in the absence of lumenal proteins, the protein folding and translocation pathways are markedly altered from the in vivo state (data not shown). In panel A, wild type, full-length preprolactin was translated in the presence or absence of native and reconstituted membranes and translocation activity assayed as protection from proteolytic degradation (Prot. K) of the signal cleaved, mature prolactin in the absence, but not the presence of detergent (CHAPS). In panel B, wild type, full-length preprolactin was translated in the presence or absence of native and reconstituted membranes and translocation activity assayed by sedimentation assays of membrane association. P, pellet (membrane) fraction; S, supernatant. p and m indicate the precursor and mature forms of the translation product. In panel C, wild type, fulllength prolactin was synthesized in the presence or absence of native and reconstituted membranes. Following translation, reactions were alkylated with 20 mM NEM, solubilized by addition of detergent, centrifuged to remove insoluble components, and the supernatant fraction analyzed by velocity sedimentation on 8 -35% sucrose gradients. Gels shown in panel C were quantitated by PhosphorImager analysis and the distribution of translation products illustrated in panel D.

DISCUSSION
Substantial progress has been made in identifying the protein components that mediate protein translocation in the mammalian ER. It is, however, presently uncertain how the process is energetically driven. Current models suggest either of two mechanisms. In one model, the direct association of the translationally active ribosome with the protein conducting channel is thought to provide a topologically restricted pathway for the nascent chain such that the nascent chain has no topological alternative other than transfer into the ER lumen, or, in the case of membrane proteins, insertion into the ER bilayer (1,2). In this model, the random, thermal motion of the nascent chain within the protein conducting channel would serve as the driving force. Alternatively, although not exclusively, protein translocation in the mammalian ER may be energetically driven in a manner similar to that proposed for translocation in yeast ER as well as for protein import into mitochondria (3,5,6,12,25). In the latter model, the free energy for transport is derived primarily through transient physical interactions of the nascent chain with lumenal, or matrix, molecular chaperones and structural alterations in the nascent chain that accompany translocation in the ER. It has been proposed that such interactions would perform a thermal ratchet function and thereby bias the random motion of the nascent chain to yield vectorial transport (3)(4)(5).
Protein folding in the ER occurs coincident with translocation, is accompanied by a significant free energy change, can be readily modified through alterations in the primary protein sequence, and occurs in an environment, the ER lumen, which is highly enriched in molecular chaperones (7, 9 -11, 42). It is with these considerations in mind that the effects of a disrupted protein folding pathway on ER translocation were investigated in vivo and in vitro. Protein folding was disrupted through mutagenesis of the folding substrate, as well as by depletion of the ER lumenal chaperones. The folding substrate used was the secretory protein preprolactin, which contains a small, disulfide-bonded loop domain at the immediate NH 2 terminus of the mature protein (28). This region of prolactin is the first to be translocated into the ER lumen and necessarily comprises the NH 2 terminus of the first folding domain. By disrupting this domain, it was predicted that the folding pathway and interaction with lumenal chaperones and protein folding enzymes would be significantly altered at an early stage of the translocation process.
In in vivo pulse-chase studies, it was observed that the mutant prolactin (FA) rapidly entered an aggregate pool comprised of mixed protein-protein and disulfide bonded complexes, and was neither secreted nor degraded (Fig. 4). Wild type preprolactin, in contrast, was efficiently secreted, with a half-time of 45 min. pPL-FA was efficiently translocated. Following homogenization of the transfected cells, and isolation of the microsomal fraction, prolactin-FA was observed to reside within the microsomal fraction (Fig. 3). From these data we conclude that although the protein folding pathway has been significantly altered, protein translocation proceeds normally. This conclusion is consistent with a number of previous observations and indicates that during passage across the ER membrane the nascent chain remains in an extended, unstructured state and likely does not enter an aggregate pool until having accessed the ER lumen (41, 59 -61).
In investigating the energetics of translocation it was considered that protein aggregation, as a thermodynamically favorable process, could contribute a driving force to protein translocation. Indeed, extremely rapid heteroaggregate formation, occurring at or prior to chain termination, has been previously reported in cells synthesizing the Semliki Forest virus proteins E1 and p62 (51). For protein-protein aggregation to directly contribute to the energetics of translocation, however, it must occur co-translationally. Through use of truncated preprolactin precursor proteins synthesized in the presence of canine pancreas rough microsomes it was observed that indeed co-translational aggregation can occur. In these experiments, truncated forms of preprolactin, previously demonstrated to comprise defined translocation intermediates (31,36,52,57), were assembled into rough microsomes and the structural state of the nascent chains subsequently determined by velocity sedimentation of a chemically alkylated, detergent-solubilized extract. Because homotypic, co-translational protein-protein interactions are unlikely to occur (7) (although there may be exceptions to this postulate (62)), it is most likely that the aggregate state observed for the truncated prolactin intermediates reflects interactions of the newly synthesized nascent chains with lumenal proteins, and/or partially folded or incompletely assembled native proteins which would remain in the microsomal vesicle lumen during tissue isolation and membrane preparation. That we have as yet been unable to identify substantial interactions with the ER lumenal proteins suggests that the latter possibility is likely.
Two additional noteworthy observations were obtained in the in vitro system. In studies with the full-length WT and FA translation products, it was observed that translocation proceeded normally, however, a significant fraction of the WT, and the majority of the FA form of prolactin, were recovered as large, heterogeneous aggregates. Thus, although the FA folding defect could be reproduced in vitro, it was also clear that in vitro the WT protein underwent substantial misfolding. Second, in lumenal protein-depleted membranes, the efficiency of translocation and folding were markedly reduced (Fig. 7). As previously reported, the loss of the lumenal chaperones had little effect on the efficiency of the early stages of translocation, i.e. translocation up to and including signal sequence cleavage (12,58). When translated in the presence of rough microsomes lacking lumenal proteins, the vast majority of the signalcleaved WT translation products were present as large aggregates. The observation that aggregated, signal-cleaved prolactin could be recovered in the supernatant fraction following sedimentation of the membranes indicates that a significant fraction of the prolactin underwent retrograde transport from the translocon and subsequent aggregation. It should also be noted that the structural state of WT prolactin, synthesized in the lumenal protein-depleted membranes, was remarkably similar to that of the folding mutant, FA, synthesized in the presence of native membranes. These data suggest that the lumenal proteins likely perform two functions: (i) through interactions with the nascent chain early in translocation, lumenal proteins may support unidirectional transport and (ii) interactions of the lumenal chaperones with the nascent chains suppress irreversible aggregation reactions and enhance the efficiency of protein folding.
The data presented herein have significant ramifications on investigations into the molecular mechanism of protein translocation. To accurately identify the molecular basis of translocation, it is critical that the experimental system accurately mimic the in vivo scenario. In many regards, the in vitro translation/translocation systems admirably achieves this goal. Analysis of the structural state of the nascent chains indicates, however, that in vitro, translocated proteins may undergo highly aberrant folding processes and accumulate as proteinprotein and disulfide-bonded aggregates. Furthermore, such disruptions in protein folding are exacerbated under conditions, such as detergent reconstitution, in which the lumenal complement of chaperones and protein folding enzymes are lost. This is especially problematic for studies of the energetic basis of translocation for which direct roles for lumenal proteins in translocation have been identified or implicated. On the basis of these data, we suggest that the criteria for accurate protein translocation be extended to include proper protein folding and/or assembly.