Turnover of the Plasma Membrane Proteins of Hepatoma Tissue Culture Cells *

The turnover of the plasma membrane proteins of hepatoma tissue culture cells was examined by three different methods-loss of polypeptides labeled in situ by lactoperoxidase-catalyzed iodination, loss of membrane polypeptides labeled with amino acid precursors, and loss from the membrane of fucoselabeled polypeptides. In both logarithmically growing and density-inhibited cells the proteins of the membrane are degraded with a half-life of about 100 hours. This is longer than the half-life of total cell protein, 50 to 60 hours, and longer than the doubling time of the cells, about 30 hours. Similar values for the rate of degradation of the membrane proteins were obtained by each of the three techniques. The same fucose-labeled polypeptides are present in the microsomal and the plasma membrane fractions of hepatoma tissue culture cells as analyzed by electrophoresis in dodecyl sulfate-acrylamide gels. But the fucose-labeled polypeptides were lost from the microsomal fraction at a faster rate than from the plasma membrane. Autoradiographic and double labeling techniques using lZsI and 1311, or [3H]leucine and [“Clleucine were used to measure the relative rates of degradation of the proteins in the plasma membrane. All of the leucine-labeled polypeptides and the iodinated polypeptides had similar rates of degradation. These results support a model for the biogenesis of the plasma menbrane in which the proteins are incorporated and removed in large structural units.

In mammalian cells essentially all proteins are undergoing turnover (l-3). That is, they are continually being degraded and replaced by new synthesis.
There is a marked heterogeneity in degradation rate and a pronounced correlation between degradation rate and molecular size of the polypeptides in the soluble fraction of rat liver (4)(5)(6). Further, heterogeneity in degradation rate and a correlation between subunit size and relative rate of degradation have been observed for the proteins of many subcellular organelles (7), including the plasma membrane (8)(9)(10)(11) Most of the chemicals used were reagent grade from commercial suppliers. Acrylamide and N,N'-dimethylbisacrylamide were obtained from Eastman.
Sodium dodecyl sulfate, dithiothreitol, and unlabeled amino acids were obtained from Sigma. "'1 and Iv11 as sodium salts were obtained from Amersham Searle and were carrier free. L- [ Fig. 2A). In nongrowing cells (Fig. 2B), the iodinated proteins are degraded at about the same rate for the first 60 hours in culture.
After 60 hours, degradation seems to stop. But  shows what happens to these polypeptides after the cells are in culture for 72 hours, relative to cells that were not in culture.
As shown in Fig. 7, all of the polypeptides in this preparation of plasma membrane had similar SH/l'C ratios, indicating similar rates of degradation. Polypeptides separated from the soluble fraction of HTC cells showed more heterogeneity in SH/l'C ratio (Fig. S) in 500 ml of fresh medium containing a lo-fold excess of unlabeled leucine. At this time the cell density was 4.6 x lo5 cells/ml, and the cells were 98% viable. The culture was then incubated at 3'7", and at the indicated time duplicate aliquots (5 ml) were removed for the determination of cell density and radioactivity. The cells in each aliquot were centrifuged, the medium was discarded, and the pellets were rinsed with saline and frozen. All the frozen pellets were broken by freeze-thaw in 0.5 ml of distilled water, and the particulate material was suspended by sonication.
Aliquots of each homogenate then were plated on filter paper and washed twice in 10% trichloroacetic acid, followed by an ethanol/ether (l/l) wash. The filter papers were then counted.
Each of the points is the average of two determinations. Thus, the 3H/14C ratios for the polypeptides in the soluble fraction (Fig. 8) are weighted in favor of the more long-lived proteins. This is apparent by comparing the ratios for the soluble and membrane proteins in Fig. 8 with the ratios obtained for total protein and membrane protein when a short pulse of amino acid is given (Table  I). Similarly, when leucine is administered for shorter intervals than 24 hours, there is both more heterogeneity in the degradation rates and faster rates of degradation of the soluble polypeptides (data not shown).
The long labeling period with leucine also would discriminate against the short-lived polypeptides in the plasma membrane (Fig. 7). But the polypeptides accessible to iodination constitute the bulk of protein in the membrane, and the iodinated polypeptides are degraded at relatively slow rates. Thus, the results in Fig. 7 confirm the studies on the The polypeptides were separated by slab acrylamide gel electrophoresis.
The gels then were fixed, dried, and autoradiographed. The autoradiographic pattern obtained from logarithmically growing cellos ( Fig. 2A) is shown in A (left), and the pattern from density-inhibited cells (Fig. 2B) in B (right).
The approximate migration distance of standard proteins of the indicated molecular weight x 10T3is shown by the numbers (right) and by the arrows (center).
Equal amounts of radioactivity were applied to each lane in A and in B. The gels were composed of a layer of 7%% acrylamide on top of a layer of 10% acrylamide.
The interface can be seen as the sharp band approximately at the center of each gel. The amount of acrylamide of each concentration is somewhat different in A and B. The difference in size reflects differential swelling which occurred during processing of the gel.
degradation of the iodinated polypeptides and show that much of the protein in the plasma membrane is degraded with similar, and probably identical, rates. Degradation of Membrane Glycoproteins-The plasma membrane of HTC cells has about 10 major glycoproteins, as detected by growing the cells in labeled fucose or labeled glucosamine (14). The same membrane components, all having molecular weights greater than 50,000, are labeled by both of the sugars. The turnover properties of these glycoproteins were examined using L-fucose as precursor.
Since little is known about the turnover of the carbohydrate moieties of glycoproteins, we first examined the kinetics of loss of fucose from total extracts of HTC cells. HTC cells were grown in the presence of L-['Hlfucose for 24 hours. At least  A plasma membrane fraction then was prepared from a mixture of the homogenates (zero time control). The other SH-labeled cells were incubated in growth medium for 72 hours at a density of 1.2 x 10B/ml. A homogenate of these cells then was prepared and frozen with the frozen homogenate of the "C-labeled cells. CloseaI circles, 3H/"C ratios of the polypeptides in the membrane fraction obtained from the latter mixture of cells. Arrow and dotted lines, mean 'H/"C ratio and one standard deviation of the mean for the polypeptides in the plasma membrane prepared from the zero time control cells; solid line, "C-labeled polypeptides in the membrane preparation separated in the 9% dodecyl sulfate-acrylamide disc gel system. 65% of the radioactivity incorporated during this interval can be recovered as fucose after acid hydrolysis of cell extracts (Table  IV). Thus, most of the precursor is present in the glycoproteins as fucose. The loss of incorporated fucose from the cells followed first order kinetics with a half-life of between 50 and 60 hours (Fig. 9). The radioactivity lost from the cells could be recovered in the medium (Fig. 9, inset). At least 85% of the radioactivity released into the medium was soluble in trichloroacetic acid, and 60% of the acid-soluble material cochromatographed with fucose in thin layer chromatography (Table  V)   At this time, aliquots of the "C-labeled cells and the 3H-laheled cells were homogenized and mixed togather. This mixture, representing the zero time control, as well as an homogenate of the other l'C-laheled cells. was immediately frozen. The other %labeled cells were suspended in complete growth medium at a concentration of 1.2 x lO'/ml. These cells were incubated at 37" for 72 hours. At the end of the incubation, the cell density was still 1.2 x 10e/ml. These 3H-laheled cells next were collected by centrifugation, washed twice with Earle's balanced salt solution, and homogenized. The homogenate was mixed and frozen with the frozen homogenate of the "C-labeled cells. A plasma membrane fraction and a microsomal fraction were prepared from this mixture of cells and from the zero time control cells. These latter cells had 'H/"C isotope ratios of 7.8 for protein in each of the cell fractions analyzed.
Arrow enclosed hy dotted lines, mean ratio and one standard deviation of the mean for the polypeptides in the plasma membrane fraction of the zero time control cells. The polypeptides were dissociated in 1% dodecyl sulfate and 1% 2-mercaptoethanol, and separated on a 9% polyacrylamide disc gel containing 0.1% dodecyl sulfate. 0, 'H/"C ratios for the fucose-labeled polypeptides in the plasma membrane fraction when the %laheled cells were in culture for 72 hours. -, "C-fucose radioactivity of the polypeptides in this fraction.
dase-catalyzed iodination, while the microsomal fraction contains little of these markers.
Consequently, the fucose-labeled glycoproteins appear to be authentic constituents of both the plasma membrane and the endoplasmic reticulum. As shown in  (Fig. lo), even more so than those of the microsomal fraction (Fig.  11). We will discuss possible reasons for this heterogeneity later, but reiterate here that while the microsomal and plasma membrane fractions may have many of the major fucose-labeled glycoproteins in com-Pm. 11. 3H/"C Ratios of fucose-labeled polypeptides in the microsomal fraction of HTC cells. Experimental details are the same as in Fig. 10, except that the polypeptides in the microsomal fraction of the cell were separated on the 9% acrylamide-dodecyl sulfate disc gel.  l), or, more directly, as measured by the loss of label from both total protein and membrane protein (Table  I). This being the case, the degradation of the HTC cell proteins which are accessible to iodination in situ can be examined.
Elsewhere (14) we showed that only plasma membrane proteins are accessible to iodination, and that these proteins represent most of the protein mass of the membrane.
The studies of Fig. 2 and Table  I show that the  membrane  proteins  accessible to iodination are being degraded exponentially at a rate that is at most one-half that of total cell protein.
The value for the half-life of the iodinated proteins obtained in Fig. 2 is somewhat longer than that which can be calculated from the data or Table  III (about  3 days). This is because in Fig. 2