- Email: [email protected]
Specific inhibition by NH4Cl of autophagy-associated proteolysis in cultured fibroblasts
Printed in Sweden Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/78/i 152.O357$02.C0/0
Experimental Cell Research 115 (1978) 357-366 SPECIFIC
INHIBITION
BY NH4CL
PROTEOLYSIS J. S. AMENTA, Department
T. J. HLIVKO,
of Pathology,
University
OF AUTOPHAGY-ASSOCIATED
IN CULTURED
FIBROBLASTS
A. G. McBEE, H. SHINOZUKA of Pittsburgh
School of Medicine,
and S. BROCHER
Pittsburgh,
PA 15261 USA
SUMMARY Rat embryo fibroblasts, prelabeled with [‘*C]leucine, showed an enhanced degradation of cell protein as well as increased peptide release when placed in a serum-deficient medium. NH&l inhibited only the induced proteolysis, but had no effect on basal protein turnover. Electron microscopy studies showed that enhanced proteolysis was associated with an increase in autophagic vacuoles containing amorphous and membranous debris, and that NH,Cl markedly increased the number of these intracellular vacuoles. Upon release from NH&l inhibition, these cells showed a compensatory enhanced release of i4C into the medium and a decrease in the number of intracellular degradative vacuoles. We conclude that enhanced proteolysis reflects an activation of the autophagic-lysosomal system in these cells and that NH&l inhibits the final hydrolysis and release steps in this mechanism.
Turnover of cellular proteins in the eukaryote appears to be effected by two distinct degradative mechanisms [ 1, 21. About the basal mechanism, we know relatively little: it hydrolyzes cell protein at a fairly constant rate of l-l.5 %/h, requires energy, can selectively degrade different proteins at different rates, and is only slowly suppressed by inhibitors of protein synthesis [3-51. An induced proteolytic mechanism (stepdown) functions primarily when the cell is suddenly subjected to a step-down nutritional state [4]. This degradative mechanism has a number of identifying characteristics: (1) it can be rapidly induced by transferring cells from a nutritionally complete growth medium to media deficient in either serum and/or amino acids; (2) the enhanced degradation is transitory and is almost immediately repressed by returning the cells to an adequate growth medium; (3) it can be rapidly inhibited by inhibitors of protein synthesis, by insulin and by 24-781816
agents inhibiting microtubular function; and (4) this degradative activity is associated with an enhanced activity of the autophagic-lysosomal system within the cell [614]. Finally, the rate of basal proteolysis correlates with cellular protease activities within the cell, whereas step-down-induced proteolysis is not associated with changes in protease activities [5, 61. Recent studies by Seglen have shown that NH&l rapidly inhibits proteolysis in isolated liver cells, dropping the rate from 5 to 1%/h [15-171. Since isolated liver cells, like cells in organ perfusion, are likely to be manifesting extremely high rates of proteolysis because of an activated step-down mechanism, this effect of NH&l on proteolysis in these cells may be, at least in part, an inhibition of the induced proteolytic mechanism. Morphologic observations on these liver cells demonstrate a rapid accumulation of vacuoles in these cells, findings consistent with the hypothesis that an Exp Cell Res 115 (1978)
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B I
3
from total 14C.At the end of the incubation cells were harvested in 0.02% EDTA (disodium salt) in phosphate-buffered saline, sonicated, and aliquots solubilized in hyamine and counted. In some additional experiments, cells were harvested by rinsing the cultures with cold phosphate-buffered saline, then overlaying the monolayer with cold 8% TCA, and scraping the flasks with a rubber policeman. Although recoveries of 14Cand 3H by this method were slightly higher, the results were not significantly different from those reported here. NH&I was added to media, as described in Results. At the concentrations used in these experiments, pH was reduced to no lower than 7.38. We have incubated tibroblasts for up to 3 days with 20 mM NH&I and find no differences in protein synthesis and growth in these cultures. Apparently NH&I is a relatively nontoxic agent to cultured fibroblasts. In experiments measuring proteolysis during recovery periods, cell monolayers were washed twice with phosphate-buffered saline at room temperature and placed in fresh growth medium for an additional 4 h. Assays for proteolysis and peptide secretion (acid-insoluble) were as previously described.
‘\‘\W----o-------d iYLIz
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I. Abscimi: NH,CI cont.; ordinate: (A) acidsoluble r4C released to medium (%/h); or (B) acidinsoluble i4C released to medium. 0, In growth medium; 0, in step-down medium. NH&I effect on degradation and release of Y-labeled cell protein. Protein in tibroblasts was labeled as described in Methods. After 24 h in non-labeled growth medium, cells were placed in either fresh growth medium or step-down medium, with NH&I as indicated on abscissa. Rate of accumulation’of total and acidsoluble i4C in medium was measured over 4 h incubations. Data from two experiments, each point represents average of six flasks. Pooled estimate of S.D. f0.21% for acid-soluble; +0.25 for acid-insoluble.
Fig.
Electron microscopy activated lysosomal-autophagic system is operating in these cells [ 181.The objectives :of this. study were to determine whether NH&l can inhibit protein degradation in [email protected] fibroblasts, to determine which -proteolytic mechanism is modified by this inhibitor, and to correlate the biochemical observations with the morphologic alterations in these cells. METHODS Cell cultures and biochemical assays ‘Detailed methods for culturing, labeling of rat embryo ftbroblasts and assaying of proteolysis rate have been previously described [I, 131.In brief, rat embryo tibroblasts from frozen stock cultures were mown in 75 cm* flasks in Eagle’s minimum essential medium supplemented with lQ% fetal calf serum (FCS) (growth medium), 0.8 &i Lji4C]leucine, and 1.0 &i [3H]thymidine. Cultures were chased for 24 h prior to the experiment with unlabeled fresh growth medium to eliminate fast turnover proteins. The rate of degradation of cell proteins was measured by assaying the amount of trichloroacetic acid (TCA)-soluble **C accumulating during I, 2 and 4 h of incubation in either growth or serum-free medium. Total i4C accumulating in the medium was measured by placing aliquots of medium into hyamine, incubating at 37°C for 1 h, and then counting in scintillation fluid. Acid-insoluble 14C in media was calculated by subtraction of soluble **C Exp Cell Res 115 (1978)
After the media was decanted, the cells were rinsed with phosphate-buffered saline three times. Two per cent glutaraldehyde in Na cacodylate buffer 0. I25 M, with sucrose 0.75 M, pH 7.4, was placed on the monolayer for 2-3 min, the cells were then scraped off with a rubber policeman into centrifuge tubes and spun at 2800 rpm in an IEC Model 5 centrifuge for 5 min. The total time in glutaraldehyde averaged about I5 min. The glutaraldehyde was replaced with Na cacodylate buffer 0. I25 M and the cells centrifuged for another I5 min to form a solid pellet. The pellet was rinsed twice more with buffer, transferred to glass vials, and fixed for 2 h in 2% 0~0,. After 30 min in OsO,, the pellets were diced. The tissue was rinsed three times with Na cacodylate buffer, dehydrated in increasing concentrations of alcohol, intiltrated with 1: I pronvlene oxide : epon overnight. and embedded the next morning. Ultrathin sect&s were cut on a Sorvall MT-I, stained with uranvl acetate and lead citrate, and examined on a Philips-200 electron microscope.
RESULTS NH&l inhibited primarily step-down induced proteolysis in cultured rat embryo fibroblasts. While fibroblasts in growth medium degraded cellular protein at a rate of 1.3%/h, fibroblasts placed in serum-free step-down medium increased protein degradation to 3.5%/h (fig. 1a). NH&l at a concentration of 5 mM reduced the proteolysis rate in cells in SD medium to 1.2 %;
Ammonia
Fig. 2. Effect of step-down medium on tibroblasts. (b) Cells incubated in step-down medium for 4 h. Di-
lated RER is prominent. Compare with control (a).
progressively higher concentrations produced little additional inhibition. In contrast, proteolysis in the control cells was relatively insensitive to NH&l, suppressing slightly from 1.3 to 0.9%/h at the highest NH&l concentration of 20 mM. In both groups of cells, NH&l appeared to suppress proteolysis to a minimum of approx. 0.9-1.0%/h. The degree of inhibition achieved at these NH,Cl concentrations duplicated the inhibitory effects observed in isolated rat liver cells [15]. These data indicated that the effects of NH&l on cellular proteolysis was not specific for liver cells, but was a general phenomenon in cultured mammalian cells, inhibiting primarily the step-down-activated degradative mechanism. We have previously reported that cultures of rat embryo fibroblasts in step-down medium release peptides to the medium, as measured by the accumulation of TCAinsoluble 14Cin the culture medium [ 11.The original observations, however, did not distinguish among at least four possible mechanisms: cell losses into the medium, disintegration of cells on the monolayer, loss of
inhibition
of protein degradation
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Arrows indicate increased vacuoles, some in proximity to Golgi, some fusing with cell membrane.
protein or cell fragments through an altered plasma membrane, and loss of protein by exocytosis from an activated autophagiclysosomal system. In these experiments, TCA-insoluble 14C was released into the culture medium at a rate of 0.8 %/h from the cells placed in fresh growth medium, while step-down medium increased this to 2.2 %/h (fig. 1b). NH,Cl inhibited the release of labeled peptide to the medium, though again this inhibitory effect was largely upon the cells in the step-down state. Although the concentrations of NH,Cl which inhibited peptide release approximated the concentrations inhibiting release of acid-soluble 14C,a notable difference was the failure of high NH,Cl concentrations to suppress peptide release to basal levels. The recovery of 3H from the fibroblast monolayers in step-down medium was approx. 64% less than the corresponding recoveries from control cultures. NH,Cl at the same concentrations which inhibited proteolysis and the release of acid-insoluble [14C]peptide had no effect upon cell loss in step-down medium. We interpreted these data to mean that step-down medium inExp Cd Res I15 ( 1978)
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Fig.
3. Effect of NH,CI on tibroblasts. Cells incubated in growth medium, containing 20 mM NH&l for 4 h. (a) Autophagic vacuoles lined by single membrane containing loose vesicular and amorphous cellular material. Some vacuoles also contained (b) densely
packed cellular material and some predominantly clear spaces with (c) little or (b) no cellular material; (d) rare vacuoles with double membrane contained packed ribosomes.
creased cell losses into the medium, adding to acid-insoluble ‘*C found in the medium. It is likely that the acid-insoluble l*C found in media with high NH&I concentrations represents a low level of either cell loss or cell disintegration, a process which is slightly enhanced in cultures placed in serum-free medium. Cells in growth medium, however, appear to release small amounts of acid-insoluble 14C,a process that is stimulated by step-down and inhibited by NH,Cl. Morphologic studies confirmed that cells in step-down medium exhibited an active
autophagic-lysosomal system and that these vacuoles were capable of releasing their contents into the culture medium. The effects of serum deprivation for 4 h were most noticeable in the endoplasmic reticulum (ER) and the vacuolar apparatus. Rough endoplasmic reticulum (RER) was frequently dilated, though it should be emphasized that only a fraction of the cells showed this response (fig. 2b). Similarly, some of the cells in step-down medium showed an increase in single membrane vacuoles containing amorphous debris,
Exp Cd RPS I IS ( 1978)
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Fig. 4. Effect of 20 mM NH&l
on fibroblasts incubated in stepdown medium for 4 h. Numerous vacuoles containing (a, b) amorphous cellular material, membranes, packed ribosomes (single arrows) and (c) large clear spaces; (d) dilated Golgi vacuoles (single ar-
rows) and vacuole releasing amorphous cellular material (double arrows). (b) Membrane whorls not in vacuoles also observed (double arrow). Dilated rough ER prominent.
small vesicular portions of membrane, and small dense particles that resembled ribosomes. Clear areas were often prominent in these vacuoles. Dense bodies and whorls were also prominent in some cells, but since such changes were also frequent in cells from control cultures in growth medium, we could not ascertain if these latter findings were quantitatively significant. Fibroblasts in fresh growth medium containing 20 mM NH&l for 4 h showed frequent changes in the vacuolar apparatus, similar but much more prominent than
those in the cells in step-down medium described in the preceding paragraph. Most commonly encountered were large vacuoles with moderately dense amorphous material (fig. 3 b) or a looser amorphous material in which vesicular membrane material was visualized (fig. 3a). Also seen in these cells were vacuoles in which a large portion of the space within the vacuole was clear (fig. 3~). Occasionally we observed a double lined vacuole, usually containing distinctive ribosomal particles in a moderately dense aggregation (fig. 3 d). Dilated endoplasmic Exp Cell Res 115 (1978)
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quently indistinct ribosome particles, vesicular membrane fragments, and amorphous material along with large clear spaces (fig. 4a, 6). Many cells were loaded with large vacuoles almost entirely devoid of stainable material (fig. 4~). Dilated Golgi vacuoles were also distinctive, along with the dilated A B C D A B C D RER. In some cells we could identify vacuoles emptying their contents into the me3 b dium (fig. 4d). This was also seen in the t cells in step-down medium without NH& but we could not ascertain from the morphologic frequency of these observations whether this differed quantitatively between the cultures in step-down medium A B C D A B C D alone and those cultures in step-down medium containing NH&I. Similar changes Fig. 5. Abscissa: (lefr) treatment period; cultures in A, growth medium; B, growth medium with NH,CI; C, were observed in cells incubated with 10 step-down medium; D, step-down medium with NH&I; (right) recovery period, same cultures placed mM NH,Cl. in growth medium; ordinate: (a) acid-soluble r4C in These morphologic observations documedium (%/h); (b) acid-insoluble ‘T in medium mented that serum-free medium produced a (%/h). (a) Cell proteolysis during and after NH&I treat- marked alteration in the vacuolar apparatus ment. Fibroblast cultures labeled as described in Methods. Incubated for 4 h in fresh growth medium of the tibroblast, inducing an acute auto(clear bar); growth medium with 20 mM NH&I (dotted phagic response, prominently affecting the bar); step-down medium (slashed bar); or step-down rough endoplasmic reticulum, ribosomes with 20 mM NH&I (slashed-dotted bar). In recovery period cells incubated for additional 4 h in fresh growth and other small membranous vacuoles (? medium. Rate of accumulation of acid-soluble i4C in smooth ER). NH,Cl did not inhibit the automedia was measured. Data from two experiments; each point represents average of six flasks. Pooled phagic response; rather, the vacuolar estimate of step-down *0.14%; (b) protein release during and after NH&I treatment. Rate of accumula- changes became striking and well-defined. tion of acid-insoluble ‘T in medium cultured by sub- We concluded that the inhibitory effects of tracting acid-soluble r*C from total IT in medium. on step-down-induced proteolysis Data points as in (a). Pooled estimate of step-down NH&l f0.18%. and protein release were not mediated by a block in the initial stages of this autophagic response, but more likely were reticulum, though occasionally seen, was not a prominent finding in these cells, con- through an inhibition in the hydrolysis of trasting with fibroblasts in the step-down the cellular material in the vacuole as well as in the release of vacuolar contents into medium. The most striking morphologic altera- the medium. NH,Cl, by blocking the final tions were produced in the fibroblasts stages of the step-down-induced proteolytic placed for 4 h in step-down medium con- mechanism, produced an accumulation of taining 20 mM NH&l. Numerous large these vacuoles within the cell and served to vacuoles with single membranes were scat- highlight the morphologic changes associtered throughout most of the cells in the ated with the induced hyperactivity of the monolayer. These contained dense but fre- autophagic-lysosomal system.
r-l
Exp Cell Res 115 (1978)
Ammonia inhibition of protein degradation The large number of vacuoles observed in the fibroblasts at the end of the 4 h incubation in either step-down medium, growth medium with NH,Cl, or step-down medium with NH,Cl raised the additional question: did these cells, loaded with autophagic vacuoles and secondary lysosomes after 4 h of blockade with NH&l, show a compensatory enhanced proteolysis and release of [14C]leucine when the cells were returned to growth medium? Fibroblasts were placed in experimental media for 4 h and then followed for an additional 4 h in fresh growth medium. The proteolysis rate in the control culture was 1.5 %/h, increased to 2.8% in the cultures placed in step-down medium (fig. 5 a); NH,Cl inhibited proteolysis in both to 1.2%/h, essentially confirming previous results. The cell cultures in the recovery growth medium all showed slightly lower rates of proteolysis, between l.O1.1 %/h, with the exception of the cultures previously incubated in step-down medium with NH,Cl. Cells in which step-down proteolysis had been inhibited by NH,Cl now showed an increase in protein degradation. These data, however, did not provide a total explanation for the fate of the material in these vacuoles. Vacuole accumulation, though of a lesser degree, was also observed in fibroblasts incubated for 4 h in step-down media (without NH,Cl) and growth medium with NH4Cl (fig. 3), yet we could not detect an increased release of acid-soluble 14Cinto the medium during the 4 h recovery period (fig. 5 a). Since the vacuoles in these fibroblasts were largely cleared during the 4 h recovery period (unpublished data), an addition release mechanism was sought. Studies on the release of [14C]peptide into the medium during the recovery period indicated that a large fraction of the accumulated cellular material was subsequently re-
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leased as acid-insoluble 14Cdirectly into the medium (fig. 5 b). In the presence of NH,Cl, the acid-insoluble 14C accumulating in the media was inhibited, confirming previous results. On recovery all cells previously in step-down medium, growth medium with NH4C1, and step-down medium with NH4Cl showed an increase in peptide release; this was particularly striking for the cultures previously in step-down medium with NH4CI. These data indicated that the vacuoles accumulating in cells treated with the three experimental media released some, and in some cases, all of the residual vacuolar contents into the medium during the recovery period. This experiment also suggested an explanation for the slight inhibition of proteolysis produced by NH,Cl in control cultures in fresh growth medium. After being placed in fresh medium, control cell cultures showed a slight increase in proteolysis rate, but returned to a lower rate of proteolysis thereafter (fig. 5a). This lower basal proteolytic rate, 1.1 %/h, was observed during the second 4 h incubation period and was maintained for at least 24 h [ 11.Cell cultures previously exposed to step-down medium or growth medium with NH4Cl also showed a similar low rate of proteolysis during the recovery period. This suggested that the slight inhibitory effect of NH,Cl on cell cultures placed in fresh growth medium at the beginning of the experimental period was on the autophagic-lysosomal system, which usually functions at a low level of activity and had been briefly activated by the change of media. NH,Cl thus appears to be a specific and rapid inhibitor of induced autophagy-mediated proteolysis. Residual proteolysis in the presence of NH,Cl in effect defines the basal proteolysis mechanism. After 4 h in recovery medium fibroblasts Exp Cell Res I I5 (1978)
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showed almost no vacuoles with amorphous and membranous debris when studied by electron microscopy. This decrease was particularly noticeable for cells previously incubated in step-down medium containing NH&I. The morphologic observations confirmed the biochemical data, indicating that fibroblasts which had accumulated vacuoles during incubation in either step-down medium or media containing NH&I released this material, either as partly hydrolyzed or acid-insoluble 14C,into the recovery growth medium. DISCUSSION These studies with rat embryo fibroblasts in culture indicate that NH,Cl is a potent and specific inhibitor of induced cellular proteolysis, resulting from activation of the autophagic-lysosomal system. Seglen’s observations in the isolated hepatocyte are consistent with this hypothesis, since an activated vacuolar apparatus has been demonstrated by electron microscopy in these cells [ 15, 171 and the concentrations of NH,Cl necessary to inhibit proteolysis are about the same for both isolated hepatocytes and fibroblasts. Use of fibroblast cultures offers the additional advantage of distinguishing between basal protein turnover in the cell and induced cell proteolysis. We have previously shown that libroblasts placed in serum-free medium activate the autophagic-lysosomal system and increase protein degradation two- to threefold [ 1, 6, 71. The step-down state in fibroblasts appears to correspond to the metabolic state of the incubated isolated hepatocytes used by Seglen, as well as the metabolic state of the liver cells in organ perfusion [12]. NH,Cl appears to be an inhibitor of this activated vacuolar system, but is virtually without effect on basal protein deExp Cell Res I I5 (1978)
gradation in these cells. Knowles & Ballard [2], using canavanine-containing proteins in hepatoma cells, have also demonstrated that NH4CI does not significantly affect the B pathway of protein degradation (synonomous with our basal degradation) but does inhibit pathway A (activated by nutritional step-down). Recently, Glimelius, Westermark & Wasteson [19] have shown that NH4Cl inhibits the turnover of sulfated glycosaminoglycans in human glial cells, turnover which occurs under basal conditions in the lysosomal system. The slight inhibition of proteolysis we observed in libroblasts placed in fresh growth medium appears to be due to a small, transitory induced proteolysis which can occur with manipulation of the cell cultures [20]. NH4Cl reduces proteolysis in all cultures to approximately the proteolysis rate of the equilibrated cells achieved during the second incubation. These data suggest that the specificity of NH4Cl inhibition is such as to be virtually synonomous with proteolysis occurring in an activated lysosomal system, while the residual proteolysis in NH4Cltreated cells defines the basal degradative mechanism. The enhanced activity of the vacuolar apparatus in these cultures also appears to be associated with an increased release of cell peptides into the medium, defined as 14Clabeled peptides precipitable in cold 8 % trichloroacetic acid (TCA). While cell loss into the medium accounts for some of the acid-insoluble 14C in the medium, the difference between the C3H]thymidine recovered in the cell monolayer from control and step-down incubations is not sufficient to account for the observed [14C]leucine in the medium. In addition, NH4Cl has no effect on loss of r3H]thymidine into the medium; its effect on protein release into the medium must be on some mechanism of protein re-
Ammonia
lease from the cell. Finally, direct observations with electron microscopy have demonstrated release of the vacuolar contents into the medium. The autophagic process induced in these cells appears to have some degree of specificity. The appearance of the vacuolar contents suggest that much of the phagocytosed material is membrane and ribosomes, i.e., most likely endoplasmic reticulum. The activated vacuolar apparatus induced by perfusion of the rat liver also appears to be loaded with both rough and smooth ER [21]. Apparently step-down medium induces an autophagic response in the cultured tibroblast, entrapping preferentially endoplasmic reticulum, which is either hydrolyzed or to some degree released in a partially degraded form into the medium. This enhanced release of acid-insoluble [14C]peptides is also inhibited by NH,Cl, apparently by inhibiting the exocytosis responsible for this peptide release. We can only speculate whether this is a direct effect on vacuolar movement in the cell, the fusion of vacuolar and plasma membrane, or whether altered pH within the secondary lysosome in some way modifies the subsequent steps in the exocytosis mechanism [18, 221. Seglen & Reith have observed a similar inhibition of protein secretion in liver cell suspensions and suggested that the enlarged protein-filled vacuoles, having a diminished mobility within the cell, are thereby delayed in their movement toward the cell membrane [23]. Returning the cells to a growth medium immediately suppresses the enhanced hydrolysis of cellular proteins, even though the cells are loaded with protein-containing vacuoles (fig. 5 a). In addition, during the recovery period this accumulated material is released largely as acid-insoluble 14Cinto the medium (fig. 5 b), although cells incubated in step-down me-
inhibition
of protein degradation
365
dium with NH4Cl appear to be an exception, showing enhanced release of 14Cin the recovery period in both acid-soluble and acidinsoluble form. Clearly hydrolysis within the secondary lysosome, as well as the release of partially degraded contents into the medium, involves control mechanisms that extend beyond the mere juxtapositioning of substrate and enzymes within the secondary lysosome. The known effect of NH,CI on lysosomal pH suggests the possibility that regulation of lysosomal pH may be part of the control mechanism regulating the hydrolysis of peptides within the secondary lysosome [22, 241. If this be the case, pH regulation within the autophagic-lysosomal vacuole must be rapidly altered under very specific cellular controls and may have effects extending beyond the activation of acid hydrolases within the vacuole. REFERENCES 1. Amenta, J S, Baccino, F M & Sargus, M J, Intracellular protein catabolism, II (ed V Turk & N Marks) p. 27. Plenum Press, New York (1977). 2. Knowles, S E Jr Ballard, F J, Biochem j 156(1976) 609. 3. Poole, B & Wibo, M, J biol them 248 (1973) 6221. 4. Hershko, A & Tomkins, G M, J biol them 246 (1971) 710. 5. Amenta, J S, Sargus, M J & Baccino, F M, Biochim biophys acta 476 (1977) 253. 6. Amenta, J S, Sargus, M J, Venkatesan, S & Shinozuko, H, J cell physiol. In press. 7. Amenta, J S, Sargus, M J & Baccino, F M, Biothem j 168(1977) 223. 8. Wibo, M & Poole, B, J cell bio163 (1975) 430. 9. Ballard, F J, Control mechanisms in cancer (ed W E Criss, T Ono & J R Sabine) p. 379. Raven Press, New York (1976). 10. Epstein, D; Eli&-Bishko, S & Hershko, A, Biochemistry 14 (1975) 5199. 11. Warburton, M J & Poole, B, Proc natl acad sci US 74 (1977) 2427. 12. Neely, A N, Nelson, P B & Mortimore, G E, Biochim bionhvs acta 338 11974)458. 13. Amenta, J S, Baccino, F M &‘Sargus, M J, Biochim biophys acta 45 1 (1976) 5 11. 14. Gunn, J M, Ballard, F J & Hanson, R W, J biol them 251 (1976) 3586. 15. Seglen, P 0, Biochem biophys res commun 66 (1975) 44. 16. - Biochim biophys acta 4% (1977) 182. Exp Cell Res II5 (1978)
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17. - Use of isolated liver cells and kidney tubules in metabolic studies (ed J M Tager, H D Soling & J R Williamson) p. 245. North-Holland Publishing Co., Amsterdam (1976). IS. Seglen, P 0 & Reith, A, Exp cell res 100 (1976) 276. 19. Glimelius, B, Westermark, B & Wasteson, A, Exp cell res I08 (1977) 23. 20. King, D W, Bensch, K G & Hill, R B, Science I31 (1960) 106. 21. Schworer, C M & Mortimore, G E, Fed proc 36 (1977) 338.
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22. Reijngoud, D J, Oud, P S, Kas, J & Tager, J M, Biochim biophys acta 448 (1976) 290. 23. Seglen, P 0 & Reith, A, Biochim biophys acta 4% (1977) 29. 24. Gordon, A H, Lysosomes in biology and pathology (ed J T Dingle) vol. 3, p. 39. North-Holland Publishing Co., Amsterdam (1973). Received December 13, 1977 Revised version received February 13, 1978 Accepted February 17, 1978
Experimental Cell Research 115 (1978) 357-366 SPECIFIC
INHIBITION
BY NH4CL
PROTEOLYSIS J. S. AMENTA, Department
T. J. HLIVKO,
of Pathology,
University
OF AUTOPHAGY-ASSOCIATED
IN CULTURED
FIBROBLASTS
A. G. McBEE, H. SHINOZUKA of Pittsburgh
School of Medicine,
and S. BROCHER
Pittsburgh,
PA 15261 USA
SUMMARY Rat embryo fibroblasts, prelabeled with [‘*C]leucine, showed an enhanced degradation of cell protein as well as increased peptide release when placed in a serum-deficient medium. NH&l inhibited only the induced proteolysis, but had no effect on basal protein turnover. Electron microscopy studies showed that enhanced proteolysis was associated with an increase in autophagic vacuoles containing amorphous and membranous debris, and that NH,Cl markedly increased the number of these intracellular vacuoles. Upon release from NH&l inhibition, these cells showed a compensatory enhanced release of i4C into the medium and a decrease in the number of intracellular degradative vacuoles. We conclude that enhanced proteolysis reflects an activation of the autophagic-lysosomal system in these cells and that NH&l inhibits the final hydrolysis and release steps in this mechanism.
Turnover of cellular proteins in the eukaryote appears to be effected by two distinct degradative mechanisms [ 1, 21. About the basal mechanism, we know relatively little: it hydrolyzes cell protein at a fairly constant rate of l-l.5 %/h, requires energy, can selectively degrade different proteins at different rates, and is only slowly suppressed by inhibitors of protein synthesis [3-51. An induced proteolytic mechanism (stepdown) functions primarily when the cell is suddenly subjected to a step-down nutritional state [4]. This degradative mechanism has a number of identifying characteristics: (1) it can be rapidly induced by transferring cells from a nutritionally complete growth medium to media deficient in either serum and/or amino acids; (2) the enhanced degradation is transitory and is almost immediately repressed by returning the cells to an adequate growth medium; (3) it can be rapidly inhibited by inhibitors of protein synthesis, by insulin and by 24-781816
agents inhibiting microtubular function; and (4) this degradative activity is associated with an enhanced activity of the autophagic-lysosomal system within the cell [614]. Finally, the rate of basal proteolysis correlates with cellular protease activities within the cell, whereas step-down-induced proteolysis is not associated with changes in protease activities [5, 61. Recent studies by Seglen have shown that NH&l rapidly inhibits proteolysis in isolated liver cells, dropping the rate from 5 to 1%/h [15-171. Since isolated liver cells, like cells in organ perfusion, are likely to be manifesting extremely high rates of proteolysis because of an activated step-down mechanism, this effect of NH&l on proteolysis in these cells may be, at least in part, an inhibition of the induced proteolytic mechanism. Morphologic observations on these liver cells demonstrate a rapid accumulation of vacuoles in these cells, findings consistent with the hypothesis that an Exp Cell Res 115 (1978)
358
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B I
3
from total 14C.At the end of the incubation cells were harvested in 0.02% EDTA (disodium salt) in phosphate-buffered saline, sonicated, and aliquots solubilized in hyamine and counted. In some additional experiments, cells were harvested by rinsing the cultures with cold phosphate-buffered saline, then overlaying the monolayer with cold 8% TCA, and scraping the flasks with a rubber policeman. Although recoveries of 14Cand 3H by this method were slightly higher, the results were not significantly different from those reported here. NH&I was added to media, as described in Results. At the concentrations used in these experiments, pH was reduced to no lower than 7.38. We have incubated tibroblasts for up to 3 days with 20 mM NH&I and find no differences in protein synthesis and growth in these cultures. Apparently NH&I is a relatively nontoxic agent to cultured fibroblasts. In experiments measuring proteolysis during recovery periods, cell monolayers were washed twice with phosphate-buffered saline at room temperature and placed in fresh growth medium for an additional 4 h. Assays for proteolysis and peptide secretion (acid-insoluble) were as previously described.
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I. Abscimi: NH,CI cont.; ordinate: (A) acidsoluble r4C released to medium (%/h); or (B) acidinsoluble i4C released to medium. 0, In growth medium; 0, in step-down medium. NH&I effect on degradation and release of Y-labeled cell protein. Protein in tibroblasts was labeled as described in Methods. After 24 h in non-labeled growth medium, cells were placed in either fresh growth medium or step-down medium, with NH&I as indicated on abscissa. Rate of accumulation’of total and acidsoluble i4C in medium was measured over 4 h incubations. Data from two experiments, each point represents average of six flasks. Pooled estimate of S.D. f0.21% for acid-soluble; +0.25 for acid-insoluble.
Fig.
Electron microscopy activated lysosomal-autophagic system is operating in these cells [ 181.The objectives :of this. study were to determine whether NH&l can inhibit protein degradation in [email protected] fibroblasts, to determine which -proteolytic mechanism is modified by this inhibitor, and to correlate the biochemical observations with the morphologic alterations in these cells. METHODS Cell cultures and biochemical assays ‘Detailed methods for culturing, labeling of rat embryo ftbroblasts and assaying of proteolysis rate have been previously described [I, 131.In brief, rat embryo tibroblasts from frozen stock cultures were mown in 75 cm* flasks in Eagle’s minimum essential medium supplemented with lQ% fetal calf serum (FCS) (growth medium), 0.8 &i Lji4C]leucine, and 1.0 &i [3H]thymidine. Cultures were chased for 24 h prior to the experiment with unlabeled fresh growth medium to eliminate fast turnover proteins. The rate of degradation of cell proteins was measured by assaying the amount of trichloroacetic acid (TCA)-soluble **C accumulating during I, 2 and 4 h of incubation in either growth or serum-free medium. Total i4C accumulating in the medium was measured by placing aliquots of medium into hyamine, incubating at 37°C for 1 h, and then counting in scintillation fluid. Acid-insoluble 14C in media was calculated by subtraction of soluble **C Exp Cell Res 115 (1978)
After the media was decanted, the cells were rinsed with phosphate-buffered saline three times. Two per cent glutaraldehyde in Na cacodylate buffer 0. I25 M, with sucrose 0.75 M, pH 7.4, was placed on the monolayer for 2-3 min, the cells were then scraped off with a rubber policeman into centrifuge tubes and spun at 2800 rpm in an IEC Model 5 centrifuge for 5 min. The total time in glutaraldehyde averaged about I5 min. The glutaraldehyde was replaced with Na cacodylate buffer 0. I25 M and the cells centrifuged for another I5 min to form a solid pellet. The pellet was rinsed twice more with buffer, transferred to glass vials, and fixed for 2 h in 2% 0~0,. After 30 min in OsO,, the pellets were diced. The tissue was rinsed three times with Na cacodylate buffer, dehydrated in increasing concentrations of alcohol, intiltrated with 1: I pronvlene oxide : epon overnight. and embedded the next morning. Ultrathin sect&s were cut on a Sorvall MT-I, stained with uranvl acetate and lead citrate, and examined on a Philips-200 electron microscope.
RESULTS NH&l inhibited primarily step-down induced proteolysis in cultured rat embryo fibroblasts. While fibroblasts in growth medium degraded cellular protein at a rate of 1.3%/h, fibroblasts placed in serum-free step-down medium increased protein degradation to 3.5%/h (fig. 1a). NH&l at a concentration of 5 mM reduced the proteolysis rate in cells in SD medium to 1.2 %;
Ammonia
Fig. 2. Effect of step-down medium on tibroblasts. (b) Cells incubated in step-down medium for 4 h. Di-
lated RER is prominent. Compare with control (a).
progressively higher concentrations produced little additional inhibition. In contrast, proteolysis in the control cells was relatively insensitive to NH&l, suppressing slightly from 1.3 to 0.9%/h at the highest NH&l concentration of 20 mM. In both groups of cells, NH&l appeared to suppress proteolysis to a minimum of approx. 0.9-1.0%/h. The degree of inhibition achieved at these NH,Cl concentrations duplicated the inhibitory effects observed in isolated rat liver cells [15]. These data indicated that the effects of NH&l on cellular proteolysis was not specific for liver cells, but was a general phenomenon in cultured mammalian cells, inhibiting primarily the step-down-activated degradative mechanism. We have previously reported that cultures of rat embryo fibroblasts in step-down medium release peptides to the medium, as measured by the accumulation of TCAinsoluble 14Cin the culture medium [ 11.The original observations, however, did not distinguish among at least four possible mechanisms: cell losses into the medium, disintegration of cells on the monolayer, loss of
inhibition
of protein degradation
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Arrows indicate increased vacuoles, some in proximity to Golgi, some fusing with cell membrane.
protein or cell fragments through an altered plasma membrane, and loss of protein by exocytosis from an activated autophagiclysosomal system. In these experiments, TCA-insoluble 14C was released into the culture medium at a rate of 0.8 %/h from the cells placed in fresh growth medium, while step-down medium increased this to 2.2 %/h (fig. 1b). NH,Cl inhibited the release of labeled peptide to the medium, though again this inhibitory effect was largely upon the cells in the step-down state. Although the concentrations of NH,Cl which inhibited peptide release approximated the concentrations inhibiting release of acid-soluble 14C,a notable difference was the failure of high NH,Cl concentrations to suppress peptide release to basal levels. The recovery of 3H from the fibroblast monolayers in step-down medium was approx. 64% less than the corresponding recoveries from control cultures. NH,Cl at the same concentrations which inhibited proteolysis and the release of acid-insoluble [14C]peptide had no effect upon cell loss in step-down medium. We interpreted these data to mean that step-down medium inExp Cd Res I15 ( 1978)
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Fig.
3. Effect of NH,CI on tibroblasts. Cells incubated in growth medium, containing 20 mM NH&l for 4 h. (a) Autophagic vacuoles lined by single membrane containing loose vesicular and amorphous cellular material. Some vacuoles also contained (b) densely
packed cellular material and some predominantly clear spaces with (c) little or (b) no cellular material; (d) rare vacuoles with double membrane contained packed ribosomes.
creased cell losses into the medium, adding to acid-insoluble ‘*C found in the medium. It is likely that the acid-insoluble l*C found in media with high NH&I concentrations represents a low level of either cell loss or cell disintegration, a process which is slightly enhanced in cultures placed in serum-free medium. Cells in growth medium, however, appear to release small amounts of acid-insoluble 14C,a process that is stimulated by step-down and inhibited by NH,Cl. Morphologic studies confirmed that cells in step-down medium exhibited an active
autophagic-lysosomal system and that these vacuoles were capable of releasing their contents into the culture medium. The effects of serum deprivation for 4 h were most noticeable in the endoplasmic reticulum (ER) and the vacuolar apparatus. Rough endoplasmic reticulum (RER) was frequently dilated, though it should be emphasized that only a fraction of the cells showed this response (fig. 2b). Similarly, some of the cells in step-down medium showed an increase in single membrane vacuoles containing amorphous debris,
Exp Cd RPS I IS ( 1978)
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361
Fig. 4. Effect of 20 mM NH&l
on fibroblasts incubated in stepdown medium for 4 h. Numerous vacuoles containing (a, b) amorphous cellular material, membranes, packed ribosomes (single arrows) and (c) large clear spaces; (d) dilated Golgi vacuoles (single ar-
rows) and vacuole releasing amorphous cellular material (double arrows). (b) Membrane whorls not in vacuoles also observed (double arrow). Dilated rough ER prominent.
small vesicular portions of membrane, and small dense particles that resembled ribosomes. Clear areas were often prominent in these vacuoles. Dense bodies and whorls were also prominent in some cells, but since such changes were also frequent in cells from control cultures in growth medium, we could not ascertain if these latter findings were quantitatively significant. Fibroblasts in fresh growth medium containing 20 mM NH&l for 4 h showed frequent changes in the vacuolar apparatus, similar but much more prominent than
those in the cells in step-down medium described in the preceding paragraph. Most commonly encountered were large vacuoles with moderately dense amorphous material (fig. 3 b) or a looser amorphous material in which vesicular membrane material was visualized (fig. 3a). Also seen in these cells were vacuoles in which a large portion of the space within the vacuole was clear (fig. 3~). Occasionally we observed a double lined vacuole, usually containing distinctive ribosomal particles in a moderately dense aggregation (fig. 3 d). Dilated endoplasmic Exp Cell Res 115 (1978)
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Amenta et al.
quently indistinct ribosome particles, vesicular membrane fragments, and amorphous material along with large clear spaces (fig. 4a, 6). Many cells were loaded with large vacuoles almost entirely devoid of stainable material (fig. 4~). Dilated Golgi vacuoles were also distinctive, along with the dilated A B C D A B C D RER. In some cells we could identify vacuoles emptying their contents into the me3 b dium (fig. 4d). This was also seen in the t cells in step-down medium without NH& but we could not ascertain from the morphologic frequency of these observations whether this differed quantitatively between the cultures in step-down medium A B C D A B C D alone and those cultures in step-down medium containing NH&I. Similar changes Fig. 5. Abscissa: (lefr) treatment period; cultures in A, growth medium; B, growth medium with NH,CI; C, were observed in cells incubated with 10 step-down medium; D, step-down medium with NH&I; (right) recovery period, same cultures placed mM NH,Cl. in growth medium; ordinate: (a) acid-soluble r4C in These morphologic observations documedium (%/h); (b) acid-insoluble ‘T in medium mented that serum-free medium produced a (%/h). (a) Cell proteolysis during and after NH&I treat- marked alteration in the vacuolar apparatus ment. Fibroblast cultures labeled as described in Methods. Incubated for 4 h in fresh growth medium of the tibroblast, inducing an acute auto(clear bar); growth medium with 20 mM NH&I (dotted phagic response, prominently affecting the bar); step-down medium (slashed bar); or step-down rough endoplasmic reticulum, ribosomes with 20 mM NH&I (slashed-dotted bar). In recovery period cells incubated for additional 4 h in fresh growth and other small membranous vacuoles (? medium. Rate of accumulation of acid-soluble i4C in smooth ER). NH,Cl did not inhibit the automedia was measured. Data from two experiments; each point represents average of six flasks. Pooled phagic response; rather, the vacuolar estimate of step-down *0.14%; (b) protein release during and after NH&I treatment. Rate of accumula- changes became striking and well-defined. tion of acid-insoluble ‘T in medium cultured by sub- We concluded that the inhibitory effects of tracting acid-soluble r*C from total IT in medium. on step-down-induced proteolysis Data points as in (a). Pooled estimate of step-down NH&l f0.18%. and protein release were not mediated by a block in the initial stages of this autophagic response, but more likely were reticulum, though occasionally seen, was not a prominent finding in these cells, con- through an inhibition in the hydrolysis of trasting with fibroblasts in the step-down the cellular material in the vacuole as well as in the release of vacuolar contents into medium. The most striking morphologic altera- the medium. NH,Cl, by blocking the final tions were produced in the fibroblasts stages of the step-down-induced proteolytic placed for 4 h in step-down medium con- mechanism, produced an accumulation of taining 20 mM NH&l. Numerous large these vacuoles within the cell and served to vacuoles with single membranes were scat- highlight the morphologic changes associtered throughout most of the cells in the ated with the induced hyperactivity of the monolayer. These contained dense but fre- autophagic-lysosomal system.
r-l
Exp Cell Res 115 (1978)
Ammonia inhibition of protein degradation The large number of vacuoles observed in the fibroblasts at the end of the 4 h incubation in either step-down medium, growth medium with NH,Cl, or step-down medium with NH,Cl raised the additional question: did these cells, loaded with autophagic vacuoles and secondary lysosomes after 4 h of blockade with NH&l, show a compensatory enhanced proteolysis and release of [14C]leucine when the cells were returned to growth medium? Fibroblasts were placed in experimental media for 4 h and then followed for an additional 4 h in fresh growth medium. The proteolysis rate in the control culture was 1.5 %/h, increased to 2.8% in the cultures placed in step-down medium (fig. 5 a); NH,Cl inhibited proteolysis in both to 1.2%/h, essentially confirming previous results. The cell cultures in the recovery growth medium all showed slightly lower rates of proteolysis, between l.O1.1 %/h, with the exception of the cultures previously incubated in step-down medium with NH,Cl. Cells in which step-down proteolysis had been inhibited by NH,Cl now showed an increase in protein degradation. These data, however, did not provide a total explanation for the fate of the material in these vacuoles. Vacuole accumulation, though of a lesser degree, was also observed in fibroblasts incubated for 4 h in step-down media (without NH,Cl) and growth medium with NH4Cl (fig. 3), yet we could not detect an increased release of acid-soluble 14Cinto the medium during the 4 h recovery period (fig. 5 a). Since the vacuoles in these fibroblasts were largely cleared during the 4 h recovery period (unpublished data), an addition release mechanism was sought. Studies on the release of [14C]peptide into the medium during the recovery period indicated that a large fraction of the accumulated cellular material was subsequently re-
363
leased as acid-insoluble 14Cdirectly into the medium (fig. 5 b). In the presence of NH,Cl, the acid-insoluble 14C accumulating in the media was inhibited, confirming previous results. On recovery all cells previously in step-down medium, growth medium with NH4C1, and step-down medium with NH4Cl showed an increase in peptide release; this was particularly striking for the cultures previously in step-down medium with NH4CI. These data indicated that the vacuoles accumulating in cells treated with the three experimental media released some, and in some cases, all of the residual vacuolar contents into the medium during the recovery period. This experiment also suggested an explanation for the slight inhibition of proteolysis produced by NH,Cl in control cultures in fresh growth medium. After being placed in fresh medium, control cell cultures showed a slight increase in proteolysis rate, but returned to a lower rate of proteolysis thereafter (fig. 5a). This lower basal proteolytic rate, 1.1 %/h, was observed during the second 4 h incubation period and was maintained for at least 24 h [ 11.Cell cultures previously exposed to step-down medium or growth medium with NH4Cl also showed a similar low rate of proteolysis during the recovery period. This suggested that the slight inhibitory effect of NH,Cl on cell cultures placed in fresh growth medium at the beginning of the experimental period was on the autophagic-lysosomal system, which usually functions at a low level of activity and had been briefly activated by the change of media. NH,Cl thus appears to be a specific and rapid inhibitor of induced autophagy-mediated proteolysis. Residual proteolysis in the presence of NH,Cl in effect defines the basal proteolysis mechanism. After 4 h in recovery medium fibroblasts Exp Cell Res I I5 (1978)
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showed almost no vacuoles with amorphous and membranous debris when studied by electron microscopy. This decrease was particularly noticeable for cells previously incubated in step-down medium containing NH&I. The morphologic observations confirmed the biochemical data, indicating that fibroblasts which had accumulated vacuoles during incubation in either step-down medium or media containing NH&I released this material, either as partly hydrolyzed or acid-insoluble 14C,into the recovery growth medium. DISCUSSION These studies with rat embryo fibroblasts in culture indicate that NH,Cl is a potent and specific inhibitor of induced cellular proteolysis, resulting from activation of the autophagic-lysosomal system. Seglen’s observations in the isolated hepatocyte are consistent with this hypothesis, since an activated vacuolar apparatus has been demonstrated by electron microscopy in these cells [ 15, 171 and the concentrations of NH,Cl necessary to inhibit proteolysis are about the same for both isolated hepatocytes and fibroblasts. Use of fibroblast cultures offers the additional advantage of distinguishing between basal protein turnover in the cell and induced cell proteolysis. We have previously shown that libroblasts placed in serum-free medium activate the autophagic-lysosomal system and increase protein degradation two- to threefold [ 1, 6, 71. The step-down state in fibroblasts appears to correspond to the metabolic state of the incubated isolated hepatocytes used by Seglen, as well as the metabolic state of the liver cells in organ perfusion [12]. NH,Cl appears to be an inhibitor of this activated vacuolar system, but is virtually without effect on basal protein deExp Cell Res I I5 (1978)
gradation in these cells. Knowles & Ballard [2], using canavanine-containing proteins in hepatoma cells, have also demonstrated that NH4CI does not significantly affect the B pathway of protein degradation (synonomous with our basal degradation) but does inhibit pathway A (activated by nutritional step-down). Recently, Glimelius, Westermark & Wasteson [19] have shown that NH4Cl inhibits the turnover of sulfated glycosaminoglycans in human glial cells, turnover which occurs under basal conditions in the lysosomal system. The slight inhibition of proteolysis we observed in libroblasts placed in fresh growth medium appears to be due to a small, transitory induced proteolysis which can occur with manipulation of the cell cultures [20]. NH4Cl reduces proteolysis in all cultures to approximately the proteolysis rate of the equilibrated cells achieved during the second incubation. These data suggest that the specificity of NH4Cl inhibition is such as to be virtually synonomous with proteolysis occurring in an activated lysosomal system, while the residual proteolysis in NH4Cltreated cells defines the basal degradative mechanism. The enhanced activity of the vacuolar apparatus in these cultures also appears to be associated with an increased release of cell peptides into the medium, defined as 14Clabeled peptides precipitable in cold 8 % trichloroacetic acid (TCA). While cell loss into the medium accounts for some of the acid-insoluble 14C in the medium, the difference between the C3H]thymidine recovered in the cell monolayer from control and step-down incubations is not sufficient to account for the observed [14C]leucine in the medium. In addition, NH4Cl has no effect on loss of r3H]thymidine into the medium; its effect on protein release into the medium must be on some mechanism of protein re-
Ammonia
lease from the cell. Finally, direct observations with electron microscopy have demonstrated release of the vacuolar contents into the medium. The autophagic process induced in these cells appears to have some degree of specificity. The appearance of the vacuolar contents suggest that much of the phagocytosed material is membrane and ribosomes, i.e., most likely endoplasmic reticulum. The activated vacuolar apparatus induced by perfusion of the rat liver also appears to be loaded with both rough and smooth ER [21]. Apparently step-down medium induces an autophagic response in the cultured tibroblast, entrapping preferentially endoplasmic reticulum, which is either hydrolyzed or to some degree released in a partially degraded form into the medium. This enhanced release of acid-insoluble [14C]peptides is also inhibited by NH,Cl, apparently by inhibiting the exocytosis responsible for this peptide release. We can only speculate whether this is a direct effect on vacuolar movement in the cell, the fusion of vacuolar and plasma membrane, or whether altered pH within the secondary lysosome in some way modifies the subsequent steps in the exocytosis mechanism [18, 221. Seglen & Reith have observed a similar inhibition of protein secretion in liver cell suspensions and suggested that the enlarged protein-filled vacuoles, having a diminished mobility within the cell, are thereby delayed in their movement toward the cell membrane [23]. Returning the cells to a growth medium immediately suppresses the enhanced hydrolysis of cellular proteins, even though the cells are loaded with protein-containing vacuoles (fig. 5 a). In addition, during the recovery period this accumulated material is released largely as acid-insoluble 14Cinto the medium (fig. 5 b), although cells incubated in step-down me-
inhibition
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dium with NH4Cl appear to be an exception, showing enhanced release of 14Cin the recovery period in both acid-soluble and acidinsoluble form. Clearly hydrolysis within the secondary lysosome, as well as the release of partially degraded contents into the medium, involves control mechanisms that extend beyond the mere juxtapositioning of substrate and enzymes within the secondary lysosome. The known effect of NH,CI on lysosomal pH suggests the possibility that regulation of lysosomal pH may be part of the control mechanism regulating the hydrolysis of peptides within the secondary lysosome [22, 241. If this be the case, pH regulation within the autophagic-lysosomal vacuole must be rapidly altered under very specific cellular controls and may have effects extending beyond the activation of acid hydrolases within the vacuole. REFERENCES 1. Amenta, J S, Baccino, F M & Sargus, M J, Intracellular protein catabolism, II (ed V Turk & N Marks) p. 27. Plenum Press, New York (1977). 2. Knowles, S E Jr Ballard, F J, Biochem j 156(1976) 609. 3. Poole, B & Wibo, M, J biol them 248 (1973) 6221. 4. Hershko, A & Tomkins, G M, J biol them 246 (1971) 710. 5. Amenta, J S, Sargus, M J & Baccino, F M, Biochim biophys acta 476 (1977) 253. 6. Amenta, J S, Sargus, M J, Venkatesan, S & Shinozuko, H, J cell physiol. In press. 7. Amenta, J S, Sargus, M J & Baccino, F M, Biothem j 168(1977) 223. 8. Wibo, M & Poole, B, J cell bio163 (1975) 430. 9. Ballard, F J, Control mechanisms in cancer (ed W E Criss, T Ono & J R Sabine) p. 379. Raven Press, New York (1976). 10. Epstein, D; Eli&-Bishko, S & Hershko, A, Biochemistry 14 (1975) 5199. 11. Warburton, M J & Poole, B, Proc natl acad sci US 74 (1977) 2427. 12. Neely, A N, Nelson, P B & Mortimore, G E, Biochim bionhvs acta 338 11974)458. 13. Amenta, J S, Baccino, F M &‘Sargus, M J, Biochim biophys acta 45 1 (1976) 5 11. 14. Gunn, J M, Ballard, F J & Hanson, R W, J biol them 251 (1976) 3586. 15. Seglen, P 0, Biochem biophys res commun 66 (1975) 44. 16. - Biochim biophys acta 4% (1977) 182. Exp Cell Res II5 (1978)
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17. - Use of isolated liver cells and kidney tubules in metabolic studies (ed J M Tager, H D Soling & J R Williamson) p. 245. North-Holland Publishing Co., Amsterdam (1976). IS. Seglen, P 0 & Reith, A, Exp cell res 100 (1976) 276. 19. Glimelius, B, Westermark, B & Wasteson, A, Exp cell res I08 (1977) 23. 20. King, D W, Bensch, K G & Hill, R B, Science I31 (1960) 106. 21. Schworer, C M & Mortimore, G E, Fed proc 36 (1977) 338.
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22. Reijngoud, D J, Oud, P S, Kas, J & Tager, J M, Biochim biophys acta 448 (1976) 290. 23. Seglen, P 0 & Reith, A, Biochim biophys acta 4% (1977) 29. 24. Gordon, A H, Lysosomes in biology and pathology (ed J T Dingle) vol. 3, p. 39. North-Holland Publishing Co., Amsterdam (1973). Received December 13, 1977 Revised version received February 13, 1978 Accepted February 17, 1978