Relative Stability of Membrane Proteins in Escherichia coli

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1 JouRNAL OF BACTEROLOGY, May 1981, p /81/ $02.00/0 Vol. 146, No. 2 Relative Stability of Membrane Proteins in Escherichia coli DEAN W. SCHROER AND ANN C. ST. JOHN* Department ofmicrobiology and Bureau ofbiological Research, Rutgers University, New Brunswick, New Jersey Received 21 November 1980/Accepted 7 February 1981 The relative stability ofmembrane proteins in Escherichia coli was investigated to determine whether these proteins are degraded at heterogeneous rates and, if so, whether the degradative rates are correlated with the sizes or charges of the proteins. Cells growing in a glucose-limited chemostat with a generation time of 15 h were labeled with [14C]leucine. After allowing 24 h for turnover of '4C-labeled proteins, the cells were labeled for 15 min with [HIleucine. By this protocol, the rapidly degraded proteins have a high ratio of 3H to 14C, whereas the stable proteins have a lower ratio. The total cell envelope fraction was collected by differential centrfugation, and the proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The relative ratio for each protein was determined by dividing its 3H/14C ratio by the 3H/14C ratio of the total membrane fraction. Although most of the 125 membrane proteins had relative ratios close to the average for the total membrane fraction, 19 varied ignificantly from this value. These differences were also observed when the order of addition of [14C]leucine and [3H]leucine was reversed. In control cultures labeled simultaneously with both isotopes, the relative ratios of these 19 proteins were similar to that of the total membrane fraction. Thirteen of these proteins had low relative ratios, which suggested that they were more stable than the average protein. An experiment in which the normal labeling procedure was followed by a 60-min chase period in the presence of excess unlabeled leucine suggested that the low relative ratios of 3 of these 13 proteins may be due to a slow post-translational modification step. Six membrane proteins had high relative ratios, which indicated that they were degraded rapidly. In contrast to the relationships found for soluble proteins in mm lian cells, there were no strong correlations between the degradative rates and either the isoelectric points or the molecular weights of membrane proteins in E. coli. Although little is known about the turnover rates of most bacterial proteins, the half-lives of a large number of mammalian proteins have been determined (5, 7, 8). In eucaryotes the degradative rates of soluble proteins are correlated with molecular weights (3-5) and isoelectric points (6) (large, acidic proteins are most labile). Some proteins, such as plasm,% membrane proteins, do not demonstrate the size and charge correlations (2, 9-11). There is considerable controversy as to whether plasma membrane proteins are degraded as a unit (2, 10, 22) or at discrete rates (3, 9, 11). Similar half-lives would be expected if large segments of the plasma membrane were degraded in the lysosomes (2, 22). In contrast, heterogeneous degradative rates would suggest that the proteins are degraded by integral membrane proteases or by selective interaction with proteases in other compartments, i.e., the extracellular space, the cytoplasm, or lysosomes. The studies reported here were designed to examine the relative stability of membrane proteins in Escherichia coli. Since bacterial cells do not contain lysosomes, degradation of membrane proteins in procaryotes must utilize integral membrane proteases or involve interactions with cytoplasmic or periplasmic proteases; Our initial studies (23) indicated that the proteins in the cell envelope fraction of E. cohi had turnover rates that were slightly lower than those of the average cell protein. In these earlier studies we examined cells grown in a glucose-limited chemostat and found that 50 to 60% of the proteins were degraded over a 72-h period. Since a large percentage of the cellular proteins were stable for up to 3 days it remained possible that membrane proteins were among a group of proteins not susceptible to degradation at all. Such a completely stable class of proteins was previously reported to be found in E. coli (19, 20). Recent studies by Mosteller et al. (16) and 476

2 VOL. 146, 1981 Larrabee and co-workers (13, 14) have characterized a number of proteins from E. coli that are degraded significantly more rapidly than the average protein. These workers found no relationship between the degradative rates of the bacterial proteins and their molecular weights or isoelectric points. Whereas the above studies identified the more labile cell proteins (13, 14, 16) and proteins that underwent post-translational modifications (16), proteins with half-lives longer than 24 h could not be distinguished from completely stable proteins. An excellent method for identifying both stable and labile proteins involves labeling them with different radioisotopic forms of the same amino acid (3, 7). Slowly growing cells are essential for the double-label protocol since the time between administration of the first and second isotope must be long enough to allow turnover of both labile and stable proteins to occur. There are a number of advantages of chemostat cultures for studies of protein degradative rates in E. coli. The addition of fresh medium can be adjusted to a very slow rate so that proteins labeled with the first isotope are not diluted out over a 24-h culturing period. In addition, the cultures have a high level of viability and, perhaps most importantly, are maintained in a steady state. Therefore, when such cells are pulse-labeled with ["4C]leucine and 24 h later are pulse-labeled with [3H]- leucine, the differences in the 3H/14C ratios in individual proteins will reflect differences in degradative rates and not changes in the synthetic rates of the proteins. The rapidly degraded proteins will have high 3H/14C ratios, whereas more stable proteins will have low 3H/14C ratios. The experiments described here used such a doublelabel protocol to examine the relative stability of membrane proteins which were separated by two-dimensional polyacrylamide gel electrophoresis. MATERIALS AND METHODS Culturing conditions. E. coli A-33 (rela+ arg tip) was grown in batch cultures at 37 C in the basal salts medium previously described (23) which was supplemented with required amino acids (60 mg/liter) and glucose (5 g/liter). Chemostat cultures were grown in basal salts medium with the glucose concentration reduced to 0.7 g/liter. A Bioflow chemostat (model C30, New Brunswick Scientific Co.) was operated under the following conditions: temperature, 37 C; aeration, 0.6 liter/min; agitation, 400 rpm. The average characteristics (+ standard deviation) of the cultures from this series of chemostats were: optical density at 550 nm = 0.72 ± 0.01; ph = 6.9; dilution rate, ± ml/h; doubling time 15.2 = h. Viability (90.5 t 1.6%) was monitored by the slide culture technique described by Postgate (21). Culture purity was monitored microscopically. STABILITY OF E. COLI MEMBRANE PROTEINS 477 Labeling of cell proteins. (i) Experimental chemostats 1 and 2. After cells reached a steady state, as monitored by maintenance of a stable culture density, proteins were labeled with 0.5 izci of ['4C]- leucine per ml (290 CI/mmol). After 24 h to allow for turnover of 14C-labeled proteins, the cells were pulselabeled with [3H]leucine (0.26 uci/ml; 58 Ci/mmol) for 7 min (chemostat 1) or 15 min (chemostat 2) and then harvested immediately. (ii) Experimental chemostats 3 and 4. To ensure that differences in ratios of isotopes in individual proteins reflect differences in protein stability and not differences in utilization of the two isotopic forms of leucine, the order of addition of radioisotopes was reversed: 1.3 uci of [3H]leucine per ml was added initially, and 24 h later the culture was pulse-labeled for 15 min with 0.13 IuCi of [14C]leucine per ml (chemostat 3) or 0.26 IsCi of [14C]leucine per ml (chemostat 4). Cells in chemostat 3 were harvested immediately after the second labeling period. In experimental chemostat 4, a 1,000-fold excess of unlabeled leucine was added to the chemostat chamber after the 15-min labeling period. The cells were incubated for an additional 1-h chase period to allow any protein modification reactions to occur. Control chemostats. In control chemostats 5 and 6, [3H]leucine (0.26,Ci/ml) and [14C]leucine (0.13,uCi/ ml) were added to the chemostat chamber simultaneously for a 15-min period before the culture was harvested. Cell breakage and fractionation. All operations were conducted at 0 to 4 C. The cells were centrifuged at 10,000 x g for 10 min and washed twice in cold buffer A (1 mm Tris-hydrochloride, 10 mm MgCl2, ph 7.4). The cells were broken by three passages through a French pressure cell at 18,000 lb/in2. The unbroken cells and debris were removed by centrifugation at 3,000 x g for 10 min. The total envelope fraction (membrane fraction) was sedimented by centrifugation at 41,000 x g for 40 min. The pellet was washed twice in buffer A and resuspended in buffer A at a protein concentration of approximately 25 to 30 mg/ ml as determined by the procedure of Lowry et al. (15). The amount of ribosomal contamination of a typical membrane preparation was determined by assaying the RNA content (12). In a sample containing 100 mg of membrane protein there was 0.9 mg of RNA, which suggested that less than 0.5% of the protein in the sample was ribosomal protein. Separation of membrane proteins by two-dimensional polyacrylamide gel electrophoresis. The procedure for solubilization of membrane proteins was that of Ames and Nikaido (1). The solubilized membrane proteins were stored at -20 C for use the same day. The two-dimensional gel electrophoretic procedure of O'Farrell (17) was used with modifications suggested by Ames and Nikaido (1). The seconddimension sodium dodecyl sulfate-polyacrylamide gel was fixed overnight in 25% isopropanol-10% acetic acid-65% water and then stained with Coomassie brilliant blue R-250. Cutting and counting of second-dimension gels. To determine the isotopic ratios of individual proteins, the stained protein spots were identified by an arbitrary numbering system and dissected from the

3 478 SCHROER AND ST. JOHN gel with a scalpel. The gel pieces were solubilized at 500C overnight with 0.5 ml of NCS tissue solubilizer (Amersham-Searle)-water (9:1) and counted in 10.0 ml of toluene-based scintillation fluid (18). Background subtraction, quench correction, and correction for crossover of 14C into the 3H channel and vice versa were performed by a computer program to determine the disintegrations per minute of each isotope. RESULTS Envelope fraction of E. coli A-33. The cell envelope of gram-negative bacteria contains two membrane layers, an innermost cytoplasmic membrane and an outer membrane containing lipopolysaccharide. The total envelope of E. coli A-33 (a K-12 strain) was collected by differential centrifugation. This fraction contained approximately 125 membrane proteins as observed by Coomassie blue staining of a two-dimensional polyacrylamide gel (Fig. 1). These proteins were identified by an arbitrary numbering system. We observed similar patterns of membrane proteins from cells grown in batch cultures or in chemostat cultures, though differences in the relative abundance of certain proteins were found. Labeling the membrane fraction. Cells growing at a steady state in a chemostat chamber were labeled with ["4C]leucine (chemostats 1 and 2) or [3H]Ieucine (chemostat 3). Our previous studies (23) showed that within 15 min, 95% of the added leucine entered the acid-precipitable fraction. Turnover of the labeled proteins occurred during the next 24 h. The cells were then pulse-labeled for 7 or 15 min with [3H]leucine (chemostats 1 and 2) or ["4C]- leucine (chemostat 3). The cultures were harvested immediately after the second pulse, the envelope fraction was prepared, and the membrane proteins were separated by two-dimensional electrophoresis. The ratio of isotopes (isotope 2/isotope 1) in each protein spot was measured. The relative stability of an individual protein was determined by dividing this ratio by the ratio of isotopes in the total membrane fraction to generate the relative ratio. Proteins with high relative ratios are labile proteins; those with low relative ratios are stable proteins. Two control chemostats (chemostats 5 and 6) measured the variability of relative ratios inherent in our double-label protocol. We added [3H]leucine and ['4C]leucine simultaneously to these control chemostats 15 min before harvesting the cells. In the envelope fraction from these control cells, 92 to 97% ofthe membrane proteins had relative ratios between 0.80 and 1.20 (Fig. 2A). The average relative ratio of each protein was measured in two independent gels from both chemostats 5 and 6. The range of these ratios was from 0.76 to J. BACTERIOL. In contrast to the near-normal distribution of relative ratios from control chemostats, the relative ratios of membrane proteins from a typical experimental chemostat displayed a heterogeneous distribution (Fig. 2B). In gel A from chemostat 2, the relative ratios ranged from 0.15 to 6.62, and only 60% of the proteins had ratios between 0.80 and Thus the membrane fraction of E. coli contained some proteins that were stable and some that were more rapidly degraded than the average protein. Relative stability of membrane proteins. The relative stability of individual membrane proteins was determined by analyzing duplicate gels from several independent chemostat cultures (Table 1). The majority of membrane proteins, including the major outer membrane component (protein 72), had relative ratios close to 1.0. A sampling of the proteins with relative ratios close to the mean ratio is shown in Table 1. Thirteen of the membrane proteins had relative ratios in the experimental chemostats that, on average, were less than 0.7, but had relative ratios near 1.0 in the control chemostats. These proteins were designated as stable or modified proteins. Of these, proteins 45, 55, 56b, and 106 had ratios less than 0.7 in all expemental chemostats. These stable or modified proteins varied greatly with respect to their isoelectric points and molecular weights (Fig. 1). Six proteins had average ratios greater than 1.2 and were designated as labile proteins. Proteins 61, 92, 93, and 100 had ratios greater than 1.3 in all experimental chemostats. The isoelectric points of the labile proteins ranged from 4.7 to 6.7. Three of these proteins were in the low molecular weight portion of the gel (Fig. 1). This might suggest that low-molecular-weight proteins in E. coli membranes are more susceptible to degradation. On the other hand, these labile proteins were interspersed among proteins with both average and low relative ratios. Modified proteins. A number of proteins in exponentially growing E. coli have been shown to undergo post-translational modifications (16). If such reactions occur in our experiments, the modifications of the membrane proteins may be slow relative to the 15-min labeling period with the second isotopic form of leucine. Therefore, the incorporation of the second isotope into such proteins will be low in the isolated envelope fraction, and a modified protein may appear in the group of proteins designated as stable components. To emine this possibility, E. coli cells in chemostat 4 were labeled in the usual manner with [3H]leucine and 24 h later with ["4C]leucine, and then the cells were incubated for an additional 60-min chase period in the presence of a

4 VOL. 146, 1981 I 68V.oo I 5 5 :e ~000 A ; 000 ~- 4 C, ( -" (: 4, IiIii a ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..B;t...; STABILITY OF E. COLI MEMBRANE PROTEINS 479 ZOV...Y>..6 *}.},.d. lp+.. %,.0: :...,..; *: : 6, 6 7 " 1 i 4Sb 221z\I3 10 r O%4u d33 z d t S.0 56 A j0 rt. I.. I 4 I V6 { ± 6.0 N. we FIG. 1. Two-dimensional electropherogram of membrane proteins fiom E. coli A-33. Proteins in the total envelope fraction were separated by isoelectric focusing (from right to left) and by electrophoresis in the presence of sodium dodecyl sulfate in the vertical dinension. The proteins in the stained gel were numbered. Proteins that uere found to be stable or modified are indicated by the numbers in squares. Proteins that were found to be labile are indicated by the circled numbers. Other numbered proteins had stabilities similar to that of total envelope fraction. SmaU circles indicate positions ofproteins that stained lightly.?.'... I ,000-fold excess of unlabeled leucine. The rela- Therefore these components would appear to be tive ratios of most membrane proteins in chem- modified proteins. ostat 4 were similar to values found in other experimental chemostats (Table 1). However, three proteins (30, 37, and 107) which had low In exponentially growing E. coli, rates of prorelative ratios in chemostats 1, 2, and 3 had tein synthesis are primarily responsible for deratios close to control values when the culture termilning the intracellular concentrations of was incubated for the 60-min chase period. proteins, with the exception of a small group of

5 480 SCHROER AND ST. JOHN J. BACTERIOL. 25 A RELATIVE RATIO FIG. 2. Frequency histogram of the relative ratios of individual membrane proteins. Relative ratios were determined by dividing the ratio of isotopes in each protein by the ratio of isotopes in the total membrane fraction. (A) Membrane proteins from control chemostat 5, gel A. (B) Membrane proteins from experimental chemostat 2, gel A. rapidly degraded components (13,14,16). Under conditions of limited growth, however, the process of protein degradation may assume a larger role in controlling protein levels. Our previous studies (23) measured the rate of protein degradation in chemostat cultures and showed that 50 to 60% of pulse-labeled proteins were degraded over a 3-day period. This suggests that the average half-life of E. coli proteins is 2 to 3 days. This value is similar to the average half-life of proteins in a number of eucaryotic systems (7, 8) and suggests that most proteins in E. coli are subject to turnover. We chose to examine the relative stability of membrane proteins for a number of reasons. (i) In mammalian cells correlations have been.-1 n found between half-lives and size (3-5) or charge (6) of soluble proteins but not plasma membrane proteins (2, 9, 11). We investigated whether such correlations exist for bacterial membrane proteins. (ii) The mechanism of turnover of membrane proteins is not weli understood. This process has been studied extensively in cultured mmrnl alian cells, which demonstrate a considerable amount of internalization and recycling of the plasma membrane components (2, 10, 11, 24). Whereas plasma membrane proteins have been reported to have similar half-lives, suggesting that membrane segments are degraded in the lysosomes (2, 10, 22), membrane proteins separated by two-dimensional electrophoresis (9) show heterogeneous degradative rates. Since B

6 VOL. 146, 1981 STABILITY OF E. COLI MEMBRANE PROTEINS 481 bacterial cells do not internalize their mem- idly in these membranes may give insight into branes, they offer a good model system to study how membrane composition is controlled. the roles of intrinsic membrane proteases or The proteins in the envelope fraction were proteases in other cell compartments (cytoplasm labeled by our double-label protocol and sepaor periplasm) in the selective degradation of rated by two-dimensional electrophoresis. If membrane proteins. (iii) Protein catabolism may membrane proteins were completely stable, the play a role in determining levels of membrane relative ratios of individual proteins would be proteins and thus in the function of membranes. similar to one another. However, the membrane The compositions and functions of the inner and proteins in experimental cultures showed a outer membranes of E. coli are distinct. The marked heterogeneity in the distribution of their characterization of proteins that turn over rap- relative ratios in comparison to the proteins TABLE 1. Relative stability of membrane proteins in E. coli A-33a Experimental chemostats Control chemo- Protein no. stats Avgb Average proteins ± ± ± ± ± ± ± ± ± ± ± ± Stable and modified proteins ± c 0.66 ± C 0.62 ± ± d 0.40 ± ± d 0.38 ± a 0.67 ± bd 0.49 ± ± ± d 0.50 ± c 0.42 ± Labile proteins ± ± ± e 4.08 ± e 5.65 ± e 1.79 ± a The relative ratio in the experimental chemostats is the ratio of isotope 2/isotope 1 for each protein, divided by the ratio of isotope 2/isotope 1 for the total proteins in the gel. In control chemostats the relative ratio is the 3H/14C ratio of each protein divided by the 3H/'4C ratio of the total proteins. Each value is the average from duplicate or triplicate gels. Average ± standard deviation of nine experimental gels. c Modified proteins with low relative ratios in chemostats 1, 2, and 3 and ratios close to the mean in chemostat 4. d Proteins with relative ratios less than 0.7 in all experimental chemostats. 'Proteins with relative ratios greater than 1.2 in all experimental chemostats.

7 482 SCHROER AND ST. JOHN from simultaneously labeled control cultures (Fig. 2). In the experimental chemostats, proteins that had relative ratios less than 0.7 were considered to be more stable than the average protein, and proteins with relative ratios greater than 1.2 were considered to turn over more rapidly than the average. It should be noted that there was a continuum of relative ratios in the experimental chemostats, indicating that the average proteins (those with relative ratios between 0.7 and 1.2) were probably a heterogeneous group. If the average membrane protein has a half-life similar to that found for total cell proteins in chemostat cultures (2 days [23]), then approximately 30% of isotope 1 originally in that protein would be released during a 24-h period. In contrast, a completely stable protein would accumulate additional isotope 1 over a 24- h period by reincorporation of radioactivity released from more labile proteins. Proteins with a very slow processing step would also have an increased accumulation of isotope 1 after 24 h. Therefore, either condition would produce a protein with a low relative ratio. We could not determine the absolute half-lives of individual proteins in these studies since we could not assess the rate of reincorporation of isotope 1 during the 24-h turnover period. Among those proteins with relative ratios less than 0.7, three proteins appeared to undergo slow post-translational modification steps. When cells labeled by our double-label protocol were incubated for a 60-min chase period in the presence of excess unlabeled leucine, the three modified proteins (30, 37, and 107) had relative ratios close to control values. The precursors to these modified proteins are not known. Proteins 30 and 37 have higher molecular weights than any of the labile proteins identified by our protocol. Therefore, the six labile proteins are not the substrates for post-translational modification steps leading to the production of 30 and 37. It is not known whether any of the labile proteins is a precursor for modified protein 107, which is a low-molecular-weight component. The proteins in this study were identified by staining with Coomassie blue rather than by autoradiography. Therefore, unless a protein precursor accumulated to such an extent that it would be visualized by staining, it would not be included in our list of membrane proteins. The proteins that had relative ratios significantly different from the mean ratio varied widely with respect to molecular weight and isoelectric point. It therefore appears that these two structural parameters are not important in determining the turnover rate of membrane proteins. These results are similar to those obtained by other investigators (13, 14, 16) who examined exponentially growing E. coli. J. BACTERIOL. The identity of the membrane proteins that differ in stability from the average protein is not known. The major outer membrane proteins 71 and 72 have relative ratios close to the mean ratio. The characterization of the membrane proteins that turn over rapidly may give additional insight into the role protein degradation plays in determining the composition and functions of bacterial membranes. ACKNOWLEDGMENTS These studies have been made possible by research grants PCM and PCM from the National Science Foundation, by Biomedical Research Support Grants from Rutgers University, and by a predoctoral Fellowship from the Charles and Johanna Busch Memorial Fund to D.W.S. LITERATURE CITED 1. Ames, G. F.-L., and K. Nikaido Two dimensional gel electrophoresis of membrane proteins. Biochemistry 15: Baumann, H., and D. Doyle Turnover of plasma membrane glycoproteins and glycolipids of hepatoma tisue culture cells. J. Biol. Chem. 253: Dellnger, P. J., and R. T. 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