Degradation of Abnormal Proteins in HeLa Cells KLAVS B. HENDIL August Krogh Institute, University of Copenhagen, 13, Uniuersitetsparken, DK 21 00, Copenhagen P,
ABSTRACT The experiments show that abnormal proteins are degraded faster than normal ones in HeLa cells. Among the fragmentary proteins made in the presence of puromycin, those with low molecular weight are least stable. Proteins made after incubation with 5-fluorouracil or in the presence of some amino acid analogues are also unstable. Breakdown of proteins made in the presence or absence of puromycin is nearly unaffected by cycloheximide and is independent of pH between 7 and 8.
Proteins in living cells are continuously broken down and resynthesized. This protein turnover is the topic for a number of recent reviews (Pine, '72; Siekevitz, '72; Goldberg and Dice, '74). The physiological significance of the protein breakdown is not known but part of it may be caused by a protmlytic scavenger system which preferentially degrades abnormal proteins. Such proteins may be formed in the cell by denaturation, mutations or ambiguities in the protein synthesis system. Certain traits about cell protein catabolism support this assumption. Thus, both denaturation and the degradation in vivo of various cellular enzymes follow first order kinetics, and the rate of both processes is decreased by addition of a suitable substrate. There is ample evidence for the existence of a proteolytic scavenger system in bacterial cells (Pine, '72; Siekevitz, '72; Goldberg and Dice, '74; Kemshead and Hipkiss, '74). In the present study I have inquired into the effects of puromycin, fluorouracil, and amino acid analogues on protein degradation in HeLa cells. The design of some of the experiments is similar to that used by Pine ('67a) and by Goldberg ('72) in studies of bacteria. The results support the theory that a proteolytic scavenger system exists in eukaryotic cells as well as in bacteria. MATERIALS AND METHODS
Lcanavanine sulphate was obtained from M a n , DL-ethionine from Nutritional Biochemicals, TES 1 and HEPES from Calbiochem and calf serum from Gibco-Biocult. D-MDMP was a gift from Dr. Robert BaxJ.
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ter, Woodstock Agricultural Research Centre, Shell Research Ltd., Sittingbourne, England. Amino acids were obtained from British Drug Houses and all other biochemicals from Sigma. L- [4,5-3H] leucine and L- [ 1-14Cl leucine were obtained from The Radiochemical Centre, Amersham, England. HeLa cells were cultivated and incubated at 37°C in 6 cm plastic petri dishes (NUNC, Roskilde, Denmark). Nearly confluent monolayer cultures were used for the experiments. The growth medium was Eagle's Minimal Essential Medium with Earle's salts, 10% calf serum, 100 pg/ml streptomycin, 100 IU/ml penicillin-G and 10 mM TES plus 10 mM HEPES instead of bicarbonate. pH was 7.5. The leucine concentration (0.4 mM) was not changed during labelling or subsequent incubation. The samples were counted in 10 ml Triton X 102-toluene scintillation cocktail (Greene et al., '68) containing 5% v/v 0.5 N HC1 and with 2,5-diphenyloxazole and 1, 4-bis-(5-phenyloxazolyl-2)-benzeneas scintillators. Experimental details are described in the appropriate figure legends. Cell viability, checked with Nigrosin (Kaltenbach et al., '58), was always better than 99 % . RESULTS
HeLa cells were labelled in growth meReceived June 3, '75. Accepted Aug. 7, '75. 1 Abbreviations. TES, N-tris(hydroxymethy1)methyl2-aminoethanesulphonicacid.HEPES, N- hydroxyethylpiperazine-N-2-ethanesulphonic acid. TCA, trichloroacetic acid. SDS, sodium dodecylsulphate. D-MDMP, D-2-(4methyl-2,6-dinitroanilino)-N-methylpropionamide. PBS, phosphate buffered saline (Dulbecco and Vogt, '54) without Ca2+ and Mg2+.
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INHIBITOR CONCENTRATION, p g / m l Fig. 1 Effect of puromycin ( O ) , cycloheximide (A) or D-MDMP (W) on 3H leucine incorporation (lower curve) and subsequent decrease in protein specific activity (upper curves). Cultures of HeLa cells were exposed to growth medium with the indicated drug concentration. Twenty minutes later the medium was replaced by 2.5 ml of the same medium with 0.4 mM 3H leucine, 2.5 Cilmol. After one hour labelling i n the presence of drugs the cells were washed by 4 x 5 ml PBS with 0.5 mM unlabelled leucine. The cells were then incubated in 5 ml growth medium without isotopes or drugs for zero to three hours. They were then washed by 4 X 5 ml PBS, suspended in 1.5 ml PBS by scraping, and frozen. Due to the time used for washing the cells the initial samples were obtained 15 minutes after the end of the labelling period. One aliquot of the thawed sample was used for Lowry ('51) protein analysis. TCA was added to another aliquot to a final concentration of 8% w/v and protein was recovered on 0.22 p m pore diameter Millipore filters. These filters were placed in scintillation vials and dissolved in 1 ml Soluene 100 (Packard) containing isopropanol (50:50 v/v) and bleached with 0.3 ml 30% hydrogen peroxide. The samples were counted in 10 ml Triton-toluene scintiIlation cocktail. Protein breakdown was calculated from the decrease i n cell specific activity which occurred during incubation in unlabelled medium.
dium with 0.4 mM 3H-leucine, 2.5 Ci/mol, for 2 to 60 minutes whereupon protein specific activity was assessed as described in the legend to figure 1. The relation between
labelling time and relative specific activity of cell protein is linear and extrapolates to time zero (not shown). The result suggests that the leucine used for incorporation at-
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Fig. 2 Electrophoretic pattern of 14C:3H radioactivity ratio (a) and 3H radioactivity (b) in total HeLa cell protein, made in the presence of 0 (A) or 20 pg/ml puromycin ( 0 ) .Protein molecular weight decreases from left to right. Cells were grown for one hour in 4 cm glass petri dishes in the presence of 20 pg/ml puromycin and either 3H leucine, 25 Cilmol, or 14C leucine, 10 Ci/mol. Total leucine concentration was 0.4 mM. Controls were grown in medium containing labelled leucine but without puromycin. After the incubation period the 14C-labelled cells were incubated for a further three hours in normal growth medium. They were then washed by 4 X 3 ml PBS with 0.5 mM unlabelled leucine and dissolved in 1 % SDS, 1% mercaptoethanol in 1 mM phosphate buffer, pH 7.5, whereas the 3H-labelled cells were washed and dissolved directly after the incubation in labelled medium. Lysates from cells labelled in the presence of the same concentration of puromycin were combined. On the following day, electrophoresis was carried out on 10% polyacrylamide gels with 0.5% SDS (Weber and Osborn, '69) and with bromphenolblue as a boundary marker. The gels were then washed overnight in 0.6 N acetic acid in 30% methanol, frozen and sliced. The slices were dried, dissolved in 0.3 ml 30% hydrogen peroxide and counted in 10 ml scintillation cocktail.
tains its equilibrium specific activity within a few minutes. The use in the succeeding experiments of unlabelled extracellular leucine in order to prevent reincorporation of intracellularly released leucine is therefore justified.
The breakdown of protein synthesized in the presence of different inhibitors of protein synthesis is shown in figure 1. Proteins made in the presence of cycloheximide or MDMP have normal stability while proteins made in the presence of puromycin
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are unstable. The decrease in lability of proteins synthesized at puromycin concentrations above 10 pg/ml may be apparent rather than real. The extensive washings of the many cell cultures used to obtain the results of figure 1 imposed an interval of 15 minutes between the end of the labelling period and the first sampling. Later studies, reported below, show that a sizeable fraction of the labile proteins are degraded within 15 minutes. Thus, the protein lability figures in figure 1 represent an underestimation for the very labile proteins. On the other hand, the specific activity used to calculate the protein catabolism (shown in figure 1) decreases, not only because of protein catabolism but also
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because of cell growth. The proteins in normal cells are therefore more stable than indicated in figure 1. Cycloheximide, MDMP, or puromycin administered during the incubation in unlabelled medium had a small inhibitory effect on the degradation of protein which had been labelled in the absence of these inhibitors (not shown). Cycloheximide and MDMP are inhibitors of peptide chain elongation (McKeehan and Hardesty, '69) and of initiation of translation (Weeks and Baxter, '72), respectively. Puromycin prematurely releases polypeptide chains from the ribosome complex and allows internal initiation of translation, thus resulting in small, defective proteins (Williamson and
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Fig. 3 Degradation of protein in normal and puromycin treated HeLa cells. Cells were ex. ) or 20 pg/ml puromycin ( 0 , @). Twenty minutes posed to growth medium with 0 (0, later the medium was replaced by 2.5 ml growth medium with the same concentration of puromycin and with 0.4 mM 3H leucine, 2.5 Cilmol. After one hour labelling, the cells were washed by 4 X 5 ml PBS with 0.5 mM unlabelled leucine. The cells were then incubated in 9 ml growth medium with 0 (empty symbols), or 20 pg/ml cycloheximide (filled symbols). One milliliter medium samples were removed at intervals from the same dish and the cells were finally dissolved in 2 ml 0.1 N NaOH-0.4% sodium deoxycholate. The samples of medium and cells were processed as described by Poole and Wibo ('73), except that the scintillation cocktail was that described in MATERIALS AND METHODS. Relative counting efficiency was assessed by internal standardization. The degradation of cellular protein was measured as the increase in TCA soluble radioactivity in the medium. It is given in percent of initially incorporated radioactivity, which is calculated as the totally recovered radioactivity less the TCA soluble radioactivity at the time of the first sampling. The first sample was taken five minutes after the end of the labelling period. Appropriate corrections were made for the volume decrease due to the sampling. In this and succeeding figures each curve is drawn through the mean from two identically treated cell cultures.
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Schweet, '65). These proteins will hereafter Proteins made in the presence of puromycin vary in molecular weight but are genbe referred to as puromycin proteins. The results suggest that the aberrant erally smaller than those from untreated puromycin proteins are preferentially de- cells (fig. 2b). The puromycin proteins with graded. This assumption is further substan- high molecular weight have essentially tiated by electrophoretic analysis of pro- normal stability and the stability seems to teins made in the presence or absence of decrease with decreasing molecular weight (fig. 2a). Probably the shortest peptide puromycin. SDS-soluble proteins from cells which chains have the least chance of folding had been incubated with 3H-labelled leu- into a stable conformation and are therecine in the presence or absence of puro- fore more susceptible to the action of a mycin were mixed with similar extracts of proteolytic scavenger system. A n alternative cells incubated with 14C-labelled leucine explanation is that the aberrant proteins under the same conditions. However, the are degraded by exopeptidases which are 1%-labelled cells had been given a further more active towards the smallest proteins 3-hour incubation in unlabelled medium because of their higher proportion of endto allow the most labile proteins to become groups per weight unit. Some variation in degraded. Rapidly degraded proteins will stability among the proteins from normal have a low 14C:3H ratio under these cir- cells is also evident from figure 2a. cumstances. The proteins were separated The time course of protein breakdown on polyacrylamide gels under conditions in 3H labelled cells was followed as the rewhere electrophoretic migration decreases lease into the medium of TCA soluble rawith increasing molecular weight (Weber dioactivity. The TCA insoluble radioactivity and Osborn, '69). The size distribution of in the medium was always very low. newly synthesized proteins, represented by In one experiment (fig. 3) the cells were the 3H radioactivity, is shown in figure 2b. labelled in medium with 0 or 20 pg/ml The stability of the proteins, reflected in puromycin and thereafter incubated in their 14C:3H ratio, is shown in figure 2a. medium with or without a concentration of
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Fig. 4 Degradation of protein in control and 5-fluorouracil treated HeLa cells. The cells were grown for 24 hours with 2.5 pg/ml thymine and with 0 (x) or 2.5 pg/ml 5-fluorouracil ( 0 ) .They were then labelled for one hour in 2.5 ml growth medium without drugs but with 0.4 mM H leucine, 2.5 Cilmol. The cells were then washed by 4 X 5 ml PBS with 0.5 mM unlabelled leucine and incubated in 9 ml normal growth medium. Samples were removed as indicated and treated as described in the legend to figure 3.
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cycloheximide (20 pglml) large enough to inhibit protein synthesis by more than 90% (compare with fig. 1). Puromycin proteins are broken down rapidly compared with normal ones. The breakdown rate in the control cells is about 18% per three hours. With comparable labelling time similar rates are found in mouse leukemia cells (Pine, '67b) and rat fibroblasts (Poole and Wibo, '73) in culture. Figure 3 also demonstrates that the blocking of protein synthesis with cycloheximide has only little effect on the protein degradation in puromycin treated as well as untreated cells. Similar effects were found when puromycin or MDMP were used instead of cycloheximide (not shown). Degradation of both normal and puromycin proteins is also unaffected by pH of the medium between 7.0 and 8.0 (not shown). 5-Fluorouracil is incorporated into RNA of eukaryotic cells, where it causes ambiguities in translation, thereby forcing the cells to make aberrant proteins (Mandel, '69). HeLa cells were grown for 24
hours in medium with 2.5 pg/ml thymine and 0 or 2.5 pg/ml 5-fluorouracil. They were then labelled and incubated in the usual way in normal medium. Cells grown in fluorouracil and thus likely to make aberrant proteins show a small but significant increase in protein turnover (fig. 4). The effect of amino acid analogues was also tested. During the labelling period arginine was replaced by canavanine, phenylalanine by pfluorophenylalanine, methionine by ethionine, or tryptophan by 5fluorotryptophan. 5-Fluorotryptophan is incorporated into protein by bacteria (Browne et al., '70) while the other three analogues are incorporated also by mammalian cells (Kruse et al., '59; Fowden, '72; Rabinovitz et al., '57). The cells were then incubated in normal medium. The effect of 5-fluorotryptophan was barely significant (not shown). All the other amino acid analogues caused synthesis of unstable proteins but to a variable degree, as shown in figure 5. The same analogues if added during the incubation in unla-
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Fig. 5 Degradation of proteins made by HeLa cells in the presence of amino acid analogues. The cells were exposed to normal growth medium (x) or to medium where the normal amino acid was substituted by ethionine (o), p-fluorophenylalanine ( 0 ) or canavanine (0. Twenty minutes later the medium was replaced by 2.5 ml of the same medium ith 0.4 mM 3H leucine, 2.5 Cilmol, and the cells labelled for one hour. The cells were then washed by 4 X 5 ml PBS with 0.5 mM unlabelled leucine and incubated in 9 ml normal growth medium. Samples were removed as indicated and treated as described in the legend to figure 3.
PROTEIN DEGRADATION
belled medium have a small inhibitory effect on the degradation of prelabelled protein. DISCUSSION
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Abnormal proteins formed by mutations in mammalian cells are also unstable. Thus mutant type hemoglobin (Adams et al., '72), temperature sensitive poliovirus protein (Garfinkle and Tershak, '72), and several missense mutants of L cell hypoxanthine-guanine phosphoribosyltransferase (Capecchi et al., '74) have increased degradation rates in the living cell. Amino acid or serum starvation enhances protein breakdown in many cell types. This enhanced degradation, but not the normal degradation, is inhibited by protein synthesis inhibitors (Hershko and Tomkins, '71; Hershko et al., '71). The degradation of abnormal proteins resembles the degradation of bulk protein in normal, well nourished cells by being insensitive to cycloheximide as shown in the present study.
The consistently low concentration of TCA insoluble radioactivity in the medium during the incubation of labelled cells, the rapidity of protein breakdown under certain conditions (fig. 3 ) and the unimpaired cell viability, all indicate that the protein breakdown is part of intracellular turnover. Reincorporation of labelled leucine which would give erroneously low breakdown figures is prevented by a rapid leucine exchange between medium and precursor pool for protein synthesis. This is supported by the relationship between labelling time and 3H incorporation, as already mentioned. In addition protein synthesis inhibitors ACKNOWLEDGMENTS have only little effect on the release of laI want to thank Dr. Per Briand for havbelled leucine from the cell, even when ing introduced me to the art of cell culture, protein degradation is fast (fig. 3). The present results show that proteins Dr. Robert Baxter for the MDMP and Dr. made in the presence of puromycin, fluo- S. 0. Andersen for valuable help during rouracil, or amino acid analogues, all of the preparation of the manuscript. which give rise to aberrant proteins, have LITERATURE CITED reduced stability in HeLa cells. Puromycin Adams, J. G., W. P. Winter, D. L. Rucknagel and has previously been shown to promote proH. H. Spencer 1972 Biosynthesis of hemoglotein breakdown in bacteria (Pine, '67a; bin Ann Arbor: Evidence for catabolic and feedGoldberg, '72; Kemshead and Hipkiss, '74), back regulation. Science, 176: 1427-1429. reticulocytes (Morris et al., '63; Baglioni Baglioni, C., B. Colombo and M. Jacobs-Lorena 1969 Chain termination: A test for a possible et al., '69; McIlhinney and Hogan, '74) explanation of thalassemia. Ann. N.Y. Acad. and HTC cells (McIlhinney and Hogan, '74). Sci., 165: 212-220. Knowles et al. ('75a) and Rabinovitz and Browne, D. T., G. L. Kenyon and G. D. Hegeman 1970 Incorporation of monofluorotryptophans Fisher ('64) have shown that hepatoma into protein during the growth of Escerichia coli. cell or reticulocyte proteins which have Biochem. Biophys. Res. Commun., 39: 13-19. incorporated amino acid analogues are Capecchi, M.R.,N. E. Capecchi, S . H. Hughes and metabolically unstable. The present results G. M. Wahl 1974 Selective degradation of abnormal proteins in mammalian tissue culture support these findings. cells. Proc. Natl. Acad. Sci. (U.S.A.), 71: 4732As shown in the present work the amino 4736. acid analogues differ in the magnitude of Dulbecco, R.,and M. Vogt 1954 Plaque formatheir effect on protein stability in HeLa tion and isolation of pure lines with poliomyelitis viruses. J. Exp. Med., 99: 167-182. cells. Similar differences are found in bacL. 1972 Fluoroamino acids and proterial cells (Pine, '67a; Goldberg, '72). Fowden, tein synthesis. In: Carbon-Fluorine Compounds. These variations in effect probably reflect C. Heidelberger, ed., CIBA Foundation Sympcdifferences in the ease with which the varisium. ous amino acid analogues are incorporated G d n k l e , B. D., and D. R. Tershak 1972 Degradation of poliovirus polypeptides in vivo. Naas well as their importance in maintaining ture new biol., 238: 206208. the structure of the proteins. Studies of Goldberg, A. L. 1972 Degradation of abnormal individual hepatoma cell enzymes syntheproteins in Escherichia coli. Proc. Natl. Acad. sized in the presence of amino acid anaSci. (U.S.A.), 69: 422426. logues have shown that heat-lability and Goldberg, A. L., and J. F. Dice 1974 Intracellul a protein degradation in mammalian and bacdegradation rate in vivo need not correlate terial cells. Ann. Rev. Biochem., 43: 835-869. with one another (Johnson and Kenney, Greene, R. C., M. S. Patterson and A. H. Estes '73; Knowles et al., '75b). 1968 Use of alkylphenol surfactants for liquid
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scintillation counting of aqueous tritium samples. Anal. Chem., 37: 2035-2037. Hershko, A,, P. Mamont, R. Shields and G. M. Tomkins 1971 “Pleiotypic response.” Nature new biol., 232; 206-211. Hershko, A,, and G. M. Tomkins 1971 Studies on the degradation of tyrosine aminotransferase in hepatoma cells in culture. J. Biol. Chem., 246: 7 10-7 14. Johnson, R. W., and F. T. Kenney 1973 Regulation of tyrosine aminotransferase in rat liver. J. Biol. Chem., 248: 45284531. Kaltenbach, J. P., M. H. Kaltenbach and W. B. Lyons 1958 Nigrosin as a dye for differentiating live and dead ascites cells. Exptl. Cell. Res., 15; 112-117. Kemshead, J. T., and A. R. Hipkiss 1974 Degradation of abnormal proteins in Escherichia coli: Relative susceptibility of canavanyl proteins and puromycin peptides in uitro. Europ. J. Biochem., 45; 535540. Knowles, S. E., J. M. Gunn, R. W. Hanson and F. J. Ballard 1975a Increased degradation rates of protein synthesized in hepatoma cells in the presence of amino acid analogues. Biochem. J., 146: 595-600. Knowles. S. E., J. M. Gunn, L. Reshef, R. W. Hanson and F. J. Ballard 1975b Properties of phosphoenolpyruvate carboxykinase (guanosine triphosphate) synthesized in hepatoma cells in the presence of amino acid analogues. Biochem. J., 146: 5 8 5 5 9 3 . Kruse, P. F., P. B. White, H. A. Carter and T. A. McCoy 1959 Incorporation ofcanavanine into protein of Walker carcinosarcoma 256 cells cultured in uitro. Cancer Res., 19: 122-125. Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275. Mandel, H. G. 1969 The incorporation of 5fluorouracil into RNA and its molecular consequences. Progress in molecular and subcellular biology, 1 : 82-135. McIlhinney, A,, and B. L. M. Hogan 1974 Rapid
degradation of puromycyl peptides in hepatoma cells and reticulocytes. FEBS letters, 40: 297401. McKeehan, W., and B. Hardesty 1969 The mechanism of cycloheximide inhibition of protein synthesis in rabbit reticulocytes. Biochem. Biophys. Res. Commun., 36: 625-630. Morris, A,, R. Arlinghaus, S. Favelukes and R. Schweet 1963 Inhibition of hemoglobin synthesis by puromycin. Biochem., 2: 1084-1090. Pine, M. J. 1967a Response of intracellular proteolysis to alteration of bacterial protein and the implications in metabolic regulation. J. Bact., 93: 1527-1533. 1967b Intracellular protein breakdown in the L1210 ascites leukemia. Cancer Res., 27: 522525. 1972 Turnover of intracellular proteins. Ann. Rev. Microbiol., 26; 103-126. Poole, B., and M. Wibo 1973 Protein degradation in cultured cells. J. Biol. Chem., 248: 62216226. Rabinovitz, M., and J. M. Fisher 1964 Characteristics of the inhibition of hemoglobin synthesis in rabbit reticulocytes by threo-a-amino-Pchlorobutyric acid. Biochim. Biophys. Acta, 91 : 31 3 3 2 2 . Rabinovitz, M., M. E. Olson and D. M. Greenberg 1957 Characteristics of the inhibition by ethionine of the incorporation of methionine into proteinsof the Ehrlich ascites carcinomain uitro. J. Biol. Chem.,227; 217-224. Siekevitz, P. 1972 The turnover of proteins and the usage of information. J. Theor. Biol., 37: 321334. Weher, E., and M. Osborn 1969 The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J. Biol. Chem., 244: 4406-4412. Weeks, D. P., and R. Baxter 1972 Specific inhibition of peptide-chain initiation by 2-(4-methyl2,6-dinitroanilino)-N-methylpropionamide. Biochem., 1 1 : 30603064. Williamson, A. R., and R. Schweet 1965 Role of the genetic message in polyribosome function. J. Mol. Biol., 1 1 : 358-372.
ABSTRACT The experiments show that abnormal proteins are degraded faster than normal ones in HeLa cells. Among the fragmentary proteins made in the presence of puromycin, those with low molecular weight are least stable. Proteins made after incubation with 5-fluorouracil or in the presence of some amino acid analogues are also unstable. Breakdown of proteins made in the presence or absence of puromycin is nearly unaffected by cycloheximide and is independent of pH between 7 and 8.
Proteins in living cells are continuously broken down and resynthesized. This protein turnover is the topic for a number of recent reviews (Pine, '72; Siekevitz, '72; Goldberg and Dice, '74). The physiological significance of the protein breakdown is not known but part of it may be caused by a protmlytic scavenger system which preferentially degrades abnormal proteins. Such proteins may be formed in the cell by denaturation, mutations or ambiguities in the protein synthesis system. Certain traits about cell protein catabolism support this assumption. Thus, both denaturation and the degradation in vivo of various cellular enzymes follow first order kinetics, and the rate of both processes is decreased by addition of a suitable substrate. There is ample evidence for the existence of a proteolytic scavenger system in bacterial cells (Pine, '72; Siekevitz, '72; Goldberg and Dice, '74; Kemshead and Hipkiss, '74). In the present study I have inquired into the effects of puromycin, fluorouracil, and amino acid analogues on protein degradation in HeLa cells. The design of some of the experiments is similar to that used by Pine ('67a) and by Goldberg ('72) in studies of bacteria. The results support the theory that a proteolytic scavenger system exists in eukaryotic cells as well as in bacteria. MATERIALS AND METHODS
Lcanavanine sulphate was obtained from M a n , DL-ethionine from Nutritional Biochemicals, TES 1 and HEPES from Calbiochem and calf serum from Gibco-Biocult. D-MDMP was a gift from Dr. Robert BaxJ.
CELL. PHYSIOL.,87: 289-296.
ter, Woodstock Agricultural Research Centre, Shell Research Ltd., Sittingbourne, England. Amino acids were obtained from British Drug Houses and all other biochemicals from Sigma. L- [4,5-3H] leucine and L- [ 1-14Cl leucine were obtained from The Radiochemical Centre, Amersham, England. HeLa cells were cultivated and incubated at 37°C in 6 cm plastic petri dishes (NUNC, Roskilde, Denmark). Nearly confluent monolayer cultures were used for the experiments. The growth medium was Eagle's Minimal Essential Medium with Earle's salts, 10% calf serum, 100 pg/ml streptomycin, 100 IU/ml penicillin-G and 10 mM TES plus 10 mM HEPES instead of bicarbonate. pH was 7.5. The leucine concentration (0.4 mM) was not changed during labelling or subsequent incubation. The samples were counted in 10 ml Triton X 102-toluene scintillation cocktail (Greene et al., '68) containing 5% v/v 0.5 N HC1 and with 2,5-diphenyloxazole and 1, 4-bis-(5-phenyloxazolyl-2)-benzeneas scintillators. Experimental details are described in the appropriate figure legends. Cell viability, checked with Nigrosin (Kaltenbach et al., '58), was always better than 99 % . RESULTS
HeLa cells were labelled in growth meReceived June 3, '75. Accepted Aug. 7, '75. 1 Abbreviations. TES, N-tris(hydroxymethy1)methyl2-aminoethanesulphonicacid.HEPES, N- hydroxyethylpiperazine-N-2-ethanesulphonic acid. TCA, trichloroacetic acid. SDS, sodium dodecylsulphate. D-MDMP, D-2-(4methyl-2,6-dinitroanilino)-N-methylpropionamide. PBS, phosphate buffered saline (Dulbecco and Vogt, '54) without Ca2+ and Mg2+.
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INHIBITOR CONCENTRATION, p g / m l Fig. 1 Effect of puromycin ( O ) , cycloheximide (A) or D-MDMP (W) on 3H leucine incorporation (lower curve) and subsequent decrease in protein specific activity (upper curves). Cultures of HeLa cells were exposed to growth medium with the indicated drug concentration. Twenty minutes later the medium was replaced by 2.5 ml of the same medium with 0.4 mM 3H leucine, 2.5 Cilmol. After one hour labelling i n the presence of drugs the cells were washed by 4 x 5 ml PBS with 0.5 mM unlabelled leucine. The cells were then incubated in 5 ml growth medium without isotopes or drugs for zero to three hours. They were then washed by 4 X 5 ml PBS, suspended in 1.5 ml PBS by scraping, and frozen. Due to the time used for washing the cells the initial samples were obtained 15 minutes after the end of the labelling period. One aliquot of the thawed sample was used for Lowry ('51) protein analysis. TCA was added to another aliquot to a final concentration of 8% w/v and protein was recovered on 0.22 p m pore diameter Millipore filters. These filters were placed in scintillation vials and dissolved in 1 ml Soluene 100 (Packard) containing isopropanol (50:50 v/v) and bleached with 0.3 ml 30% hydrogen peroxide. The samples were counted in 10 ml Triton-toluene scintiIlation cocktail. Protein breakdown was calculated from the decrease i n cell specific activity which occurred during incubation in unlabelled medium.
dium with 0.4 mM 3H-leucine, 2.5 Ci/mol, for 2 to 60 minutes whereupon protein specific activity was assessed as described in the legend to figure 1. The relation between
labelling time and relative specific activity of cell protein is linear and extrapolates to time zero (not shown). The result suggests that the leucine used for incorporation at-
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Fig. 2 Electrophoretic pattern of 14C:3H radioactivity ratio (a) and 3H radioactivity (b) in total HeLa cell protein, made in the presence of 0 (A) or 20 pg/ml puromycin ( 0 ) .Protein molecular weight decreases from left to right. Cells were grown for one hour in 4 cm glass petri dishes in the presence of 20 pg/ml puromycin and either 3H leucine, 25 Cilmol, or 14C leucine, 10 Ci/mol. Total leucine concentration was 0.4 mM. Controls were grown in medium containing labelled leucine but without puromycin. After the incubation period the 14C-labelled cells were incubated for a further three hours in normal growth medium. They were then washed by 4 X 3 ml PBS with 0.5 mM unlabelled leucine and dissolved in 1 % SDS, 1% mercaptoethanol in 1 mM phosphate buffer, pH 7.5, whereas the 3H-labelled cells were washed and dissolved directly after the incubation in labelled medium. Lysates from cells labelled in the presence of the same concentration of puromycin were combined. On the following day, electrophoresis was carried out on 10% polyacrylamide gels with 0.5% SDS (Weber and Osborn, '69) and with bromphenolblue as a boundary marker. The gels were then washed overnight in 0.6 N acetic acid in 30% methanol, frozen and sliced. The slices were dried, dissolved in 0.3 ml 30% hydrogen peroxide and counted in 10 ml scintillation cocktail.
tains its equilibrium specific activity within a few minutes. The use in the succeeding experiments of unlabelled extracellular leucine in order to prevent reincorporation of intracellularly released leucine is therefore justified.
The breakdown of protein synthesized in the presence of different inhibitors of protein synthesis is shown in figure 1. Proteins made in the presence of cycloheximide or MDMP have normal stability while proteins made in the presence of puromycin
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are unstable. The decrease in lability of proteins synthesized at puromycin concentrations above 10 pg/ml may be apparent rather than real. The extensive washings of the many cell cultures used to obtain the results of figure 1 imposed an interval of 15 minutes between the end of the labelling period and the first sampling. Later studies, reported below, show that a sizeable fraction of the labile proteins are degraded within 15 minutes. Thus, the protein lability figures in figure 1 represent an underestimation for the very labile proteins. On the other hand, the specific activity used to calculate the protein catabolism (shown in figure 1) decreases, not only because of protein catabolism but also
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because of cell growth. The proteins in normal cells are therefore more stable than indicated in figure 1. Cycloheximide, MDMP, or puromycin administered during the incubation in unlabelled medium had a small inhibitory effect on the degradation of protein which had been labelled in the absence of these inhibitors (not shown). Cycloheximide and MDMP are inhibitors of peptide chain elongation (McKeehan and Hardesty, '69) and of initiation of translation (Weeks and Baxter, '72), respectively. Puromycin prematurely releases polypeptide chains from the ribosome complex and allows internal initiation of translation, thus resulting in small, defective proteins (Williamson and
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Fig. 3 Degradation of protein in normal and puromycin treated HeLa cells. Cells were ex. ) or 20 pg/ml puromycin ( 0 , @). Twenty minutes posed to growth medium with 0 (0, later the medium was replaced by 2.5 ml growth medium with the same concentration of puromycin and with 0.4 mM 3H leucine, 2.5 Cilmol. After one hour labelling, the cells were washed by 4 X 5 ml PBS with 0.5 mM unlabelled leucine. The cells were then incubated in 9 ml growth medium with 0 (empty symbols), or 20 pg/ml cycloheximide (filled symbols). One milliliter medium samples were removed at intervals from the same dish and the cells were finally dissolved in 2 ml 0.1 N NaOH-0.4% sodium deoxycholate. The samples of medium and cells were processed as described by Poole and Wibo ('73), except that the scintillation cocktail was that described in MATERIALS AND METHODS. Relative counting efficiency was assessed by internal standardization. The degradation of cellular protein was measured as the increase in TCA soluble radioactivity in the medium. It is given in percent of initially incorporated radioactivity, which is calculated as the totally recovered radioactivity less the TCA soluble radioactivity at the time of the first sampling. The first sample was taken five minutes after the end of the labelling period. Appropriate corrections were made for the volume decrease due to the sampling. In this and succeeding figures each curve is drawn through the mean from two identically treated cell cultures.
293
PROTEIN DEGRADATION
Schweet, '65). These proteins will hereafter Proteins made in the presence of puromycin vary in molecular weight but are genbe referred to as puromycin proteins. The results suggest that the aberrant erally smaller than those from untreated puromycin proteins are preferentially de- cells (fig. 2b). The puromycin proteins with graded. This assumption is further substan- high molecular weight have essentially tiated by electrophoretic analysis of pro- normal stability and the stability seems to teins made in the presence or absence of decrease with decreasing molecular weight (fig. 2a). Probably the shortest peptide puromycin. SDS-soluble proteins from cells which chains have the least chance of folding had been incubated with 3H-labelled leu- into a stable conformation and are therecine in the presence or absence of puro- fore more susceptible to the action of a mycin were mixed with similar extracts of proteolytic scavenger system. A n alternative cells incubated with 14C-labelled leucine explanation is that the aberrant proteins under the same conditions. However, the are degraded by exopeptidases which are 1%-labelled cells had been given a further more active towards the smallest proteins 3-hour incubation in unlabelled medium because of their higher proportion of endto allow the most labile proteins to become groups per weight unit. Some variation in degraded. Rapidly degraded proteins will stability among the proteins from normal have a low 14C:3H ratio under these cir- cells is also evident from figure 2a. cumstances. The proteins were separated The time course of protein breakdown on polyacrylamide gels under conditions in 3H labelled cells was followed as the rewhere electrophoretic migration decreases lease into the medium of TCA soluble rawith increasing molecular weight (Weber dioactivity. The TCA insoluble radioactivity and Osborn, '69). The size distribution of in the medium was always very low. newly synthesized proteins, represented by In one experiment (fig. 3) the cells were the 3H radioactivity, is shown in figure 2b. labelled in medium with 0 or 20 pg/ml The stability of the proteins, reflected in puromycin and thereafter incubated in their 14C:3H ratio, is shown in figure 2a. medium with or without a concentration of
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Fig. 4 Degradation of protein in control and 5-fluorouracil treated HeLa cells. The cells were grown for 24 hours with 2.5 pg/ml thymine and with 0 (x) or 2.5 pg/ml 5-fluorouracil ( 0 ) .They were then labelled for one hour in 2.5 ml growth medium without drugs but with 0.4 mM H leucine, 2.5 Cilmol. The cells were then washed by 4 X 5 ml PBS with 0.5 mM unlabelled leucine and incubated in 9 ml normal growth medium. Samples were removed as indicated and treated as described in the legend to figure 3.
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cycloheximide (20 pglml) large enough to inhibit protein synthesis by more than 90% (compare with fig. 1). Puromycin proteins are broken down rapidly compared with normal ones. The breakdown rate in the control cells is about 18% per three hours. With comparable labelling time similar rates are found in mouse leukemia cells (Pine, '67b) and rat fibroblasts (Poole and Wibo, '73) in culture. Figure 3 also demonstrates that the blocking of protein synthesis with cycloheximide has only little effect on the protein degradation in puromycin treated as well as untreated cells. Similar effects were found when puromycin or MDMP were used instead of cycloheximide (not shown). Degradation of both normal and puromycin proteins is also unaffected by pH of the medium between 7.0 and 8.0 (not shown). 5-Fluorouracil is incorporated into RNA of eukaryotic cells, where it causes ambiguities in translation, thereby forcing the cells to make aberrant proteins (Mandel, '69). HeLa cells were grown for 24
hours in medium with 2.5 pg/ml thymine and 0 or 2.5 pg/ml 5-fluorouracil. They were then labelled and incubated in the usual way in normal medium. Cells grown in fluorouracil and thus likely to make aberrant proteins show a small but significant increase in protein turnover (fig. 4). The effect of amino acid analogues was also tested. During the labelling period arginine was replaced by canavanine, phenylalanine by pfluorophenylalanine, methionine by ethionine, or tryptophan by 5fluorotryptophan. 5-Fluorotryptophan is incorporated into protein by bacteria (Browne et al., '70) while the other three analogues are incorporated also by mammalian cells (Kruse et al., '59; Fowden, '72; Rabinovitz et al., '57). The cells were then incubated in normal medium. The effect of 5-fluorotryptophan was barely significant (not shown). All the other amino acid analogues caused synthesis of unstable proteins but to a variable degree, as shown in figure 5. The same analogues if added during the incubation in unla-
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Fig. 5 Degradation of proteins made by HeLa cells in the presence of amino acid analogues. The cells were exposed to normal growth medium (x) or to medium where the normal amino acid was substituted by ethionine (o), p-fluorophenylalanine ( 0 ) or canavanine (0. Twenty minutes later the medium was replaced by 2.5 ml of the same medium ith 0.4 mM 3H leucine, 2.5 Cilmol, and the cells labelled for one hour. The cells were then washed by 4 X 5 ml PBS with 0.5 mM unlabelled leucine and incubated in 9 ml normal growth medium. Samples were removed as indicated and treated as described in the legend to figure 3.
PROTEIN DEGRADATION
belled medium have a small inhibitory effect on the degradation of prelabelled protein. DISCUSSION
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Abnormal proteins formed by mutations in mammalian cells are also unstable. Thus mutant type hemoglobin (Adams et al., '72), temperature sensitive poliovirus protein (Garfinkle and Tershak, '72), and several missense mutants of L cell hypoxanthine-guanine phosphoribosyltransferase (Capecchi et al., '74) have increased degradation rates in the living cell. Amino acid or serum starvation enhances protein breakdown in many cell types. This enhanced degradation, but not the normal degradation, is inhibited by protein synthesis inhibitors (Hershko and Tomkins, '71; Hershko et al., '71). The degradation of abnormal proteins resembles the degradation of bulk protein in normal, well nourished cells by being insensitive to cycloheximide as shown in the present study.
The consistently low concentration of TCA insoluble radioactivity in the medium during the incubation of labelled cells, the rapidity of protein breakdown under certain conditions (fig. 3 ) and the unimpaired cell viability, all indicate that the protein breakdown is part of intracellular turnover. Reincorporation of labelled leucine which would give erroneously low breakdown figures is prevented by a rapid leucine exchange between medium and precursor pool for protein synthesis. This is supported by the relationship between labelling time and 3H incorporation, as already mentioned. In addition protein synthesis inhibitors ACKNOWLEDGMENTS have only little effect on the release of laI want to thank Dr. Per Briand for havbelled leucine from the cell, even when ing introduced me to the art of cell culture, protein degradation is fast (fig. 3). The present results show that proteins Dr. Robert Baxter for the MDMP and Dr. made in the presence of puromycin, fluo- S. 0. Andersen for valuable help during rouracil, or amino acid analogues, all of the preparation of the manuscript. which give rise to aberrant proteins, have LITERATURE CITED reduced stability in HeLa cells. Puromycin Adams, J. G., W. P. Winter, D. L. Rucknagel and has previously been shown to promote proH. H. Spencer 1972 Biosynthesis of hemoglotein breakdown in bacteria (Pine, '67a; bin Ann Arbor: Evidence for catabolic and feedGoldberg, '72; Kemshead and Hipkiss, '74), back regulation. Science, 176: 1427-1429. reticulocytes (Morris et al., '63; Baglioni Baglioni, C., B. Colombo and M. Jacobs-Lorena 1969 Chain termination: A test for a possible et al., '69; McIlhinney and Hogan, '74) explanation of thalassemia. Ann. N.Y. Acad. and HTC cells (McIlhinney and Hogan, '74). Sci., 165: 212-220. Knowles et al. ('75a) and Rabinovitz and Browne, D. T., G. L. Kenyon and G. D. Hegeman 1970 Incorporation of monofluorotryptophans Fisher ('64) have shown that hepatoma into protein during the growth of Escerichia coli. cell or reticulocyte proteins which have Biochem. Biophys. Res. Commun., 39: 13-19. incorporated amino acid analogues are Capecchi, M.R.,N. E. Capecchi, S . H. Hughes and metabolically unstable. The present results G. M. Wahl 1974 Selective degradation of abnormal proteins in mammalian tissue culture support these findings. cells. Proc. Natl. Acad. Sci. (U.S.A.), 71: 4732As shown in the present work the amino 4736. acid analogues differ in the magnitude of Dulbecco, R.,and M. Vogt 1954 Plaque formatheir effect on protein stability in HeLa tion and isolation of pure lines with poliomyelitis viruses. J. Exp. Med., 99: 167-182. cells. Similar differences are found in bacL. 1972 Fluoroamino acids and proterial cells (Pine, '67a; Goldberg, '72). Fowden, tein synthesis. In: Carbon-Fluorine Compounds. These variations in effect probably reflect C. Heidelberger, ed., CIBA Foundation Sympcdifferences in the ease with which the varisium. ous amino acid analogues are incorporated G d n k l e , B. D., and D. R. Tershak 1972 Degradation of poliovirus polypeptides in vivo. Naas well as their importance in maintaining ture new biol., 238: 206208. the structure of the proteins. Studies of Goldberg, A. L. 1972 Degradation of abnormal individual hepatoma cell enzymes syntheproteins in Escherichia coli. Proc. Natl. Acad. sized in the presence of amino acid anaSci. (U.S.A.), 69: 422426. logues have shown that heat-lability and Goldberg, A. L., and J. F. Dice 1974 Intracellul a protein degradation in mammalian and bacdegradation rate in vivo need not correlate terial cells. Ann. Rev. Biochem., 43: 835-869. with one another (Johnson and Kenney, Greene, R. C., M. S. Patterson and A. H. Estes '73; Knowles et al., '75b). 1968 Use of alkylphenol surfactants for liquid
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scintillation counting of aqueous tritium samples. Anal. Chem., 37: 2035-2037. Hershko, A,, P. Mamont, R. Shields and G. M. Tomkins 1971 “Pleiotypic response.” Nature new biol., 232; 206-211. Hershko, A,, and G. M. Tomkins 1971 Studies on the degradation of tyrosine aminotransferase in hepatoma cells in culture. J. Biol. Chem., 246: 7 10-7 14. Johnson, R. W., and F. T. Kenney 1973 Regulation of tyrosine aminotransferase in rat liver. J. Biol. Chem., 248: 45284531. Kaltenbach, J. P., M. H. Kaltenbach and W. B. Lyons 1958 Nigrosin as a dye for differentiating live and dead ascites cells. Exptl. Cell. Res., 15; 112-117. Kemshead, J. T., and A. R. Hipkiss 1974 Degradation of abnormal proteins in Escherichia coli: Relative susceptibility of canavanyl proteins and puromycin peptides in uitro. Europ. J. Biochem., 45; 535540. Knowles, S. E., J. M. Gunn, R. W. Hanson and F. J. Ballard 1975a Increased degradation rates of protein synthesized in hepatoma cells in the presence of amino acid analogues. Biochem. J., 146: 595-600. Knowles. S. E., J. M. Gunn, L. Reshef, R. W. Hanson and F. J. Ballard 1975b Properties of phosphoenolpyruvate carboxykinase (guanosine triphosphate) synthesized in hepatoma cells in the presence of amino acid analogues. Biochem. J., 146: 5 8 5 5 9 3 . Kruse, P. F., P. B. White, H. A. Carter and T. A. McCoy 1959 Incorporation ofcanavanine into protein of Walker carcinosarcoma 256 cells cultured in uitro. Cancer Res., 19: 122-125. Lowry, 0. H., N. J. Rosebrough, A. L. Farr and R. J. Randall 1951 Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275. Mandel, H. G. 1969 The incorporation of 5fluorouracil into RNA and its molecular consequences. Progress in molecular and subcellular biology, 1 : 82-135. McIlhinney, A,, and B. L. M. Hogan 1974 Rapid
degradation of puromycyl peptides in hepatoma cells and reticulocytes. FEBS letters, 40: 297401. McKeehan, W., and B. Hardesty 1969 The mechanism of cycloheximide inhibition of protein synthesis in rabbit reticulocytes. Biochem. Biophys. Res. Commun., 36: 625-630. Morris, A,, R. Arlinghaus, S. Favelukes and R. Schweet 1963 Inhibition of hemoglobin synthesis by puromycin. Biochem., 2: 1084-1090. Pine, M. J. 1967a Response of intracellular proteolysis to alteration of bacterial protein and the implications in metabolic regulation. J. Bact., 93: 1527-1533. 1967b Intracellular protein breakdown in the L1210 ascites leukemia. Cancer Res., 27: 522525. 1972 Turnover of intracellular proteins. Ann. Rev. Microbiol., 26; 103-126. Poole, B., and M. Wibo 1973 Protein degradation in cultured cells. J. Biol. Chem., 248: 62216226. Rabinovitz, M., and J. M. Fisher 1964 Characteristics of the inhibition of hemoglobin synthesis in rabbit reticulocytes by threo-a-amino-Pchlorobutyric acid. Biochim. Biophys. Acta, 91 : 31 3 3 2 2 . Rabinovitz, M., M. E. Olson and D. M. Greenberg 1957 Characteristics of the inhibition by ethionine of the incorporation of methionine into proteinsof the Ehrlich ascites carcinomain uitro. J. Biol. Chem.,227; 217-224. Siekevitz, P. 1972 The turnover of proteins and the usage of information. J. Theor. Biol., 37: 321334. Weher, E., and M. Osborn 1969 The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. J. Biol. Chem., 244: 4406-4412. Weeks, D. P., and R. Baxter 1972 Specific inhibition of peptide-chain initiation by 2-(4-methyl2,6-dinitroanilino)-N-methylpropionamide. Biochem., 1 1 : 30603064. Williamson, A. R., and R. Schweet 1965 Role of the genetic message in polyribosome function. J. Mol. Biol., 1 1 : 358-372.
