Escherichia coli. which are unable to accumulate ppgpp under such conditions (2, 28, 31). Furthermore, the

Transcription

1 JOURNAL OF BACTERIOLOGY, Sept. 1980, p /80/ /11$02.00/0 Vol. 143, No. 3 Effects of Starvation for Potassium and Other Inorganic Ions on Protein Degradation and Ribonucleic Acid Synthesis in Escherichia coli ANN C. ST. JOHN'* AND ALFRED L. GOLDBERG2 Department ofmicrobiology, Bureau of Biological Research, Rutgers University, New Brunswick, New Jersey 08903,' and Physiology Department, Harvard Medical School, Boston, Massachusetts Starvation of Escherichia coli for potassium, phosphate, or magnesium ions leads to a reversible increase in the rate of protein degradation and an inhibition ofribonucleic acid (RNA) synthesis. In cells deprived of potassium, the breakdown of the more stable cell proteins increased two- to threefold, whereas the hydrolysis of short-lived proteins, both normal ones and analog-containing polypeptides, did not change. The mechanisms initiating the enhancement of proteolysis during starvation for these ions were examined. Upon starvation for amino acids or amino acyl-transfer RNA (trna), protein breakdown increases in rea+ (but not rea) cells as a result of the rapid synthesis of guanosine-5'-diphosphate-3'- diphosphate (ppgpp). However, a lack of amino acyl-trna does not appear to be responsible for the increased protein breakdown in cells starved for inorganic ions, since protein breakdown increased in the absence of these ions in both rela + and rea cultures, and since a large excess of amino acids did not affect this response. In bacteria in which energy production is restricted, ppgpp levels also rise, and protein breakdown increases. The ion-deprived cultures did show a 40 to 75% reduction in adenosine-5'-triphosphate levels, similar to that seen upon glucose starvation. However, this decrease in ATP content does not appear to cause the increase in protein breakdown or lead to an accumulation of ppgpp. No consistent change in intracellular ppgpp levels was found in rea + or rea cells starved for these ions. In addition, in reix mutants, removal of these ions led to accelerated protein degradation even though relx cells are unable to increase ppgpp levels or proteolysis when deprived of' a carbon source. In the potassium-, phosphate-, and magnesium-deprived cultures, the addition of choramphenicol or tetracycline caused a reduction in protein breakdown toward basal levels. Such findings, however, do not indicate that protein synthesis is essential for the enhancement of protein degradation, since blockage of protein synthesis by inactivation of a temperature-sensitive valyl-trna synthetase did not restore protein catabolism to basal levels. These various results and related studies suggest that the mechanism for increased protein catabolism on starvation for inorganic ions differs from that occurring upon amino acid or carbon deprivation and probably involves an enhanced susceptibility of various cell proteins (especially ribosomal proteins) to proteolysis. In growing bacteria the average rates of protein breakdown are relatively low, but when such cells are deprived of a carbon source, nitrogen, or an essential amino acid, the degradation of cell proteins increases severalfold (11, 17, 24). It appears likely that the stimulation of proteolysis is of value to the starving cell by providing a source of amino acids for further protein synthesis or for energy metabolism (11, 12). A large variety of evidence indicates that this response requires an accumulation of guanosine-5'-diphosphate-3'-diphosphate (ppgpp) or some closely related compound in the cells. During 1223 starvation for an amino acid (11, 24, 26, 30) or amino acyl-trna (9, 28) protein degradation increases in rea+ cells, but not rela mutants, which are unable to accumulate ppgpp under such conditions (2, 28, 31). Furthermore, the actual increase in protein catabolism under these conditions is proportional to the increase in ppgpp levels (31). In addition, when cells are starved for a carbon source (29, 30, 31), or when production of ATP is restricted (29), protein catabolism increases in both rel4+ and rela strains. Treatment of amino acid-deprived or energy-depleted cells with agents that block

2 1224 ST. JOHN AND GOLDBERG ppgpp synthesis, such as tetracycline, leads to a rapid return of the rate of proteolysis to basal levels (29, 31). Finally, the rate of fall in proteolysis under these conditions depends upon the rate of disappearance of ppgpp (31). Protein catabolism increases not only in response to starvation for carbon or nitrogen sources, but also when cells are starved for inorganic nutrients, such as phosphate (26, 34) or magnesium (34). The physiological significance of this cellular response is not clear, since enhanced proteolysis should not relieve the underlying nutrient deficiency in the ion-starved cultures. It is also not known whether the lack of inorganic ions affects the degradation of the same proteins as does glucose or amino acid deprivation. To learn more about the mechanisms regulating protein breakdown in E. coli, we have systematically studied the response to starvation for various essential ions. Protein catabolism was found to increase not only during deprivation for phosphate and magnesium but also when cultures lack potassium (11). Since starvation for all these inorganic nutrients resulted in a similar increase in protein breakdown and a simultaneous decrease in RNA synthesis, a common cellular mechanism may be responsible for these effects. For example, it was suggested (11) that the coordinate changes in proteolysis and RNA synthesis may be signaled by mechanisms similar to those occurring upon energy stepdown, i.e., by a fall in cellular ATP levels and a subsequent increase in ppgpp. The present studies examine more closely the regulation of protein catabolism during ion starvation and the role of ATP levels and guanosine-bis-diphosphate in this process. MATERLU1S AND METHODS Bacterial strains and media. Escherichia coli strains A-33 reua+ arg trpa and A-33 rela arg trpa were kindly provided by B. Davis. E. coli strains NF859 metb arga reua+ reix+, NF859X metb rea relx+, and NF1035 metb rela relx were kindly provided by J. Gallant. E coli mutants NF536 leu vals(ts) reu+ and NF537 leu vals(ts) rela were kindly provided by J. D. Friesen. Cells were grown with aeration at 37 C in either the basal salts medium described previously (9) or in Tris medium (ph 7.4) containing (per liter): 7.26 g of Tris, 2.4 g of (NH4)2SO4, g of MgSO4. 7H20, g of KCI, g of Na2HPO4.7H20, and g of NaCI. Glucose (5 g/liter) and required amino acids (30 mg/ liter) were sterilized separately. To prepare starvation media, the following changes in the composition of Tris medium were made: for potassium-free, KCI was omitted; for magnesium-free, MgSO4 7H20 was omitted; for phosphate-free, Na2HPO4 *7H20 was omitted; and for nitrogen-free, (NH4)2SO4 and required amino J. BACTERIOL. acids were omitted. Growth was estimated by measuring turbidity of the cell suspension with a Klett-Summerson colorimeter (green filter) or a Gilford spectrophotometer at 550 nm. An optical density at 550 nm (OD5r,m) of 1.0 was equivalent to 95 Klett units and 4 x 108 cells/ml. Measurement of protein breakdown. Rates of protein breakdown in strains A-33, NF859, NF859X, and NF1035 were measured in cells that had grown for two generations in Tris medium containing L-4,5- [3H]leucine (0.1 uci/ml). The cells were collected on Millipore filters and washed four times with starvation medium containing 120 Lg of unlabeled leucine per ml. The cells were suspended at a density of 2 x 108 to 3 x 10' cells/ml in medium containing 120,ug of unlabeled leucine per ml. At various times, 1.0-ml samples were removed and added to 0.1 ml of 50% trichloroacetic acid. Degradation of protein was measured from the release of [3H]leucine into acid-soluble form as described previously (9). Each value is the average of two determinations which generally agreed within 5%. In strains NF536 and NF537, the breakdown of proteins labeled with ['Hlphenylalanine (0.2 PCi/ml) was measured as described previously (28). To measure the breakdown of amino acid analog-containing protein, strain A-33 rela + cells were washed two times in arginine-free medium containing 2.1,uCi of ['IC]- canavanine per ml. After 19 min, the cells were washed in ion-free medium and suspended at a density of 3 x 108 cells/ml in medium containing 120 ug of arginine per ml. The release of ['4C]canavanine into trichloroacetic acid-soluble form was measured as described previously (28). Measurement of net RNA synthesis. RNA synthesis was measured by the incorporation of 2- ['4C]uracil into material precipitable with 5% trichloroacetic acid which could be hydrolyzed by treatment with 1.0 N KOH for 16 h at 370C as described previously (29). The growth medium was supplemented with ['4C]uracil (0.05,uCi/ml, specific activity 0.56 uci/,umol). RNA content was also determined colorimetrically with the orcinol reaction (16). Measurement of nucleotides. The levels of ATP were measured after extracting the cells with 5% trichloroacetic acid. The acid was extracted with four volumes of ether, and ATP was measured in duplicate by the firefly luciferase assay as described by Stanley and Williams (27). Lyophilized firefly lanterns were obtained from Sigma Chemical Co (St. Louis, Mo.). To measure ppgpp levels, cells were collected on Millipore filters and washed four times with the lowphosphate Tris medium previously described (29) lacking one of the essential nutrients as described in the experimental results. Cells were resuspended in the same medium at a density of 1 x 108 cells/ml. A portion of the culture was used to determine cell turbidity (ODm%), and another portion was supplemented with 100,uCi of 32P per ml. Levels of ppgpp were estimated by the method of Cashel (1). RESULTS Effect of starvation for potassium on protein degradation and RNA synthesis in reia+ cells. During starvation for a source of

3 VOL. 143, 1980 carbon, nitrogen, or amino acids, E. coli cells increase their degradation of preexistent proteins, apparently to provide a source of amino acids for protein synthesis. These cells also decrease the synthesis of rrna and trna. The effects of potassium starvation on protein breakdown and RNA synthesis were therefore examined in E. coli A-33 rema. In a typical experiment, growing cells were transferred to medium lacking potassium ions, at which point growth slowed and then stopped within 60 min. The rate of breakdown of preexistent proteins increased 2- to 2.5-fold upon suspension of the cells in potassium-free medium (Fig. 1). When potassium was resupplied to the starved cells, growth resumed and protein breakdown returned to basal levels (Fig. 1). The increase in protein catabolism in potassium-deprived cells probably did not result from a secondary requirement for potassium for the biosynthesis of certain amino acids or the transport of other ions, such as phosphate. Accordingly, the addition of either a complete mixture of amino acids or a 250-fold increase in the molarity of phosphate did not reduce protein degradation or permit growth to resume. During potassium starvation, RNA synthesis was greatly reduced as measured by the incorporation of radioactive uracil (Table 1). Such changes do not reflect alterations in uracil uptake since similar decreases were seen when RNA accumulation was measured colorimetrically. The regulation of protein degradation during starvation for carbon, nitrogen or amino acids 10 z Refed K MINUTES FIG. 1. Effect ofpotassium deprivation on the rate of protein breakdown in strain A-33 reua +. Protein breakdown was measured in complete medium (O) or in medium lacking potassium (-). At the time indicated by the arrow, 4 mm KCl was added to the culture in potassium-free medium (0). PROTEIN BREAKDOWN IN E. COLI 1225 TABLE 1. Effect of starvation on protein breakdown and net RNA synthesis in E. coli A-33 rela + and A- 33 relaa A-33 rela+ A-33 rela Uracil Uracil Medium Protein incor- Protein incorbreak- pora- break- poradown tion (% down tion (% (%/h) con- (%/h) control) trol) Complete Arg, - trp N, - arg, trp - Glucose K P Mg 'To measure protein breakdown, cells were grown for two generations in complete medium containing ['Hjleucine (0.1 AtCi/ml). Cells were collected on membrane filters (Millipore Corp., Bedford, Mass.) and washed four times with starvation medium containing 120 ug of unlabeled leucine per ml. The labeled cells were suspended in complete medium or in media lacking the indicated nutrient(s). Protein breakdown was determined as described in the text. Similar results were obtained in four separate experiments. To measure net RNA synthesis, growing cells were collected on membrane filters (Millipore Corp.) and washed four times with starvation medium containing 10 jig of uracil per ml. The cells were suspended in complete medium or media lacking the indicated nutrient(s) supplemented with [14C]uracil (0.05,uCi/ml, specific activity 0.56 MCi/,umol). Incorporation of ['4C]uracil was determined as described in the text and expressed as a percentage of the incorporation found in complete medium. Similar results were found in two independent experiments. involves a preferential stimulation of the degradation of more stable polypeptides (11, 24). Normal proteins that have short half-lives or proteins with abnormal structures are degraded at a rapid rate in the presence or absence of required nutrients. We examined whether the stimulation of protein catabolism in potassiumstarved cultures also involved a selective degradation of normally stable components. Because the half-lives of cellular proteins are heterogeneous, the exposure of cells to radioactive amino acids for various periods of time will preferentially label groups of proteins having different stabilities (11). E. coli A-33 rela+ were exposed to [3H]leucine for a short period (5 min) to enrich for radioactivity in the more labile fraction of proteins. The culture was divided, and protein breakdown was immediately measured in one-half of the culture in the presence and absence of potassium (Fig. 2A). During the sub-

4 1226 ST. JOHN AND GOLDBERG J. BACTERIOL. S z 3:~ ~ ~ ~ CNTROL 4-2 CONTROL MINUTES FIG. 2. Effects ofpotassium deprivation on the degradation of the more labile recently synthesizedprotein fraction and the more stable protein fraction in E. coli A-33 rela +. Cells at a density of 5 x 108 cells/ml were exposed to 0.2,uCi of [3HJleucine per ml for 5 min. (A) A portion of the culture was washed with potassiumfree medium and suspended in complete medium (0) or in medium lacking potassium (0) to measure degradation of the more rapidly degraded fraction of cell proteins; (B) a second portion of the culture was washed and suspended in complete medium containing 120 mm unlabeled leucine. After 60 min, the cells were washed with potassium-free medium and suspended in complete medium (0) or medium lacking potassium (0) to measure the degradation of more stable proteins. sequent 60 min, the degradation of labile components was primarily responsible for the observed rate of catabolism, and the rates of protein breakdown were similar in the presence and absence of potassium. The second half of the labeled culture was allowed to grow in complete medium containing excess unlabeled leucine for 60 min to allow for the breakdown of labile components. After this treatment, protein breakdown of this culture was measured in the presence and absence of potassium (Fig. 2B). A threefold increase in the catabolism of labeled proteins was seen in the cells deprived of potassium. To examine the effect of potassium starvation on the degradation of abnormal proteins, E. coli A-33 rea+ were labeled in medium in which arginine was replaced by the arginine analog ['4C]canavanine. After transfer to medium containing arginine, the breakdown of the proteins containing ["Cicanavanine was followed in the presence and absence of potassium. As shown in Fig. 3, the rates of catabolism of these abnormal proteins were very similar in both cultures. Therefore, like other types of starvation, potassium deprivation selectively stimulates the breakdown of the more stable cell proteins. Starvation for other inorganic nutrients. z 4 w w I- at PROTEINS CONTAINING 14C-CANAVANINE MINUTES FIG. 3. Effect of potassium deprivation on the breakdown of abnormal proteins in E. coli A-33 rea. Degradation of [''Cicanavanine-containing proteins was measured in complete medium (0) and in medium lacking potassium (0).

5 VOL. 143, 1980 Phosphate or magnesium starvation also caused a two- to threefold stimulation in protein degradation (Table 1). The rate of proteolysis under these conditions was similar to that found in cells starved for glucose or nitrogen. This enhanced rate of proteolysis may be the maximal possible rate of protein catabolism since starvation for more than one required nutrient did not lead to additive increases in proteolysis: protein degradation was 5% per h in nitrogen and amino acid-free medium, 4.8% per h in potassium-free medium, and 5.5% per h in medium lacking all three nutrients. Starvation for phosphate and magnesium also led to a marked decrease in RNA synthesis, as had previously been reported (18, 26). Effect of starvation for inorganic nutrients in relaxed cells. Cells that have a defect in the stringent control system (rela) are unable to increase protein degradation or reduce RNA synthesis in response to starvation for amino acids or amino acyl-trna (9, 28, 30) (Table 1). However, both these strains show the normal increase in protein breakdown and decrease in RNA synthesis when deprived of a carbon source (29, 30) (Table 1) or when energy production is reduced (29). Likewise, in E. coli A-33 rea, the rate of protein catabolism increased twofold and RNA synthesis dropped 10-fold in the absence of a nitrogen source. Decreased RNA synthesis upon nitrogen starvation has been seen in other rela strains (15). It should be emphasized that nitrogen deprivation is not identical to amino acid starvation. In cells lacking a nitrogen source, the availability of nitrogenous bases also decreases and, in related studies, purine or pyrimidine deprivation has been shown to accelerate proteolysis in rela strains by a mechanism that appears to be independent of levels of ppgpp (Goldberg and Rosenthal, unpublished data). The effect of starvation for inorganic nutrients was examined in E. coli A- 33 rea to determine whether the regulation of proteolysis resembles that during starvation for a carbon source or starvation for amino acids. Protein catabolism was stimulated approximately twofold by the removal of glucose, nitrogen, potassium, phosphate, or magnesium (Table 1). Conditions in which catabolism was increased also led to a large decrease in the rate of RNA synthesis. Protein degradation in reix cells. Since protein degradation and RNA synthesis appear to change coordinately during a number of starvation conditions (11; Table 1), it is attractive to suggest that a common mechanism regulates both processes. Although there is an excellent correlation between the levels of ppgpp and the PROTEIN BREAKDOWN IN E. COLI 1227 rates of RNA synthesis (2, 7) and protein breakdown (28) when amino acyl-trna is limiting, the kinetics of ppgpp accumulation do not correlate completely with rates of RNA synthesis during carbon starvation and certain other conditions (2, 14). To test for a possible role of ppgpp in regulating proteolysis during starvation for glucose or inorganic nutrients, we examined a mutant (rela relx) recently described by Pao and Gallant (22). This strain has a very low basal level of ppgpp and is unable to accumulate this nucleotide during starvation for glucose as a result of the reix mutation. The rates of protein degradation were measured in the isogenic series, NF859 (rea+ relk+), NF859X (reu relx+), and NF1035 (rela reix) during incubation in complete medium or in media either lacking glucose or the required amino acid methionine (Table 2). As expected, starvation for methionine caused a twofold stimulation of proteolysis in E. coli NF859 but not in the reu strains, which do not accumulate ppgpp in the absence of required amino acids. During glucose starvation the relx+ strains showed a two- to threefold enhancement in the rate of protein degradation, whereas mutant NF1035 showed no such stimulation of protein breakdown in accord with earlier data (31). To examine whether the regulation of protein catabolism during starvation for inorganic ions was dependent on the reix gene product, strain NF859, NF859X, or NF1035 was placed in medium lacking potassium, phosphate, or magnesium ions. All three strains were able to increase protein catabolism severalfold (Table 2). Thus, the stimulation of protein catabolism during ion starvation occurs normally in the absence of functional rela and relx gene products and differs from that seen in energy-restricted cells. Levels of ATP and ppgpp in ion-starved cells. The coordinate changes in protein catabolism and RNA synthesis during potassium, magnesium, or phosphate starvation of E. coli A-33 rela + and A-33 rela cells (Table 1) suggest that a common metabolic signal may regulate these processes. It has previously been shown that a moderate inhibition (30 to 50%) of the cell's ability to generate ATP leads to a stimulation of protein catabolism as well as a reduction in growth and RNA synthesis in both reu + and rea strains. Therefore, the effect of starvation for inorganic ions on cellular ATP levels was examined (Table 3). Starvation for ions or for glucose led to a 40 to 75% decrease in ATP content of the cells. Deprivation for a source of potassium or magnesium led to a decrease in ATP similar to that induced by concentrations of respiratory inhibitors (KCN or NaNA), that

6 1228 ST. JOHN AND GOLDBERG TABLE 2. Effect of various types of starvation on protein breakdown in rela and rea rely strainsa Protein breakdown (%/2 h) Experi- Meiu ment Mulum NF859 NF859X NF1035 (rela+ (rela (rela relx+) rely') relx) 1 Complete Glucose Methio nine 2 Complete Mg Complete P Complete K a Rates of protein degradation were measured in the usual fashion. The indicated strains were grown in the presence of [3H]leucine for two generations to label cell proteins. The cells were harvested and washed four times with starvation medium supplemented with 120,ug of unlabeled leucine per ml. The cells were suspended in a complete medium or in medium lacking either glucose, methionine (a required amino acid), phosphate, or potassium. Similar results were obtained in two separate experiments. caused a twofold increase in protein catabolism. Phosphate starvation led to a 75% decrease in ATP, which was similar to the reduction found in glucose-starved cells. During carbon starvation or inhibition of energy production, an accumulation of ppgpp occurs in both rel + and rela cells as a result of a reduced rate of degradation of this nucleotide (2, 3, 28). We therefore examined whether the enhancement of protein degradation in the ionstarved cells was the result of an increase in ppgpp levels that might result from the fall in cellular ATP content. The ppgpp levels of E. coli A-33 rea+ and A-33 rel were measured during starvation for potassium and magnesium ions (Table 4). Upon starvation for amino acids, the levels of ppgpp increased seven- to eightfold in the rel + strain but did not change in the rel cells. In potassium-starved E. coli A-33 reua, the ppgpp content was 2.5- to 9-fold greater than in control cells. In the rea cells, however, no significant increase in ppgpp was found. When rel+ or rel cells were deprived of magnesium, no consistent increase above the basal ppgpp level was found, although there were considerable variations in the levels of guanosine-bis-diphosphate. Thus, the stimulation of protein degradation in potassium-starved rea cells and magnesium-starved cells (unlike that J. BACTERIOL. seen on glucose or amino acid starvation) does not appear to require an accumulation of ppgpp. Effect of chloramphenicol and tetracycline. The stimulation of protein catabolism during glucose, nitrogen, or amino acid starvation can be reversed by the addition of the antibiotics chloramphenicol or tetracycline (11, 17, 24). As shown in Fig. 4, the addition of tetracycline also lowered the rate of protein breakdown towards basal levels in cultures starved for potassium or phosphate. Similar reductions in protein breakdown were found upon addition of chloramphenicol to cells starved for inorganic ions or nitrogen (Table 5). These inhibitors interact with the ribosome and thereby block protein synthesis. Consequently, they induce a marked fall in the intracellular concentration of ppgpp because they indirectly permit intracellular pools of amino acid and RNA to increase in the starved cells (trickle-charging) (2). In addition, tetracycline directly inhibits the "stringent factor" which catalyzes the transfer TABLE 3. Levels ofatp in E. coli A-33 reua during starvation for inorganic nutrientsa Medium ATP (% control) Complete Glucose K P m e a Cells were collected on membrane filters (Millipore Corp.) and washed four times with the various media. Cells were suspended in the indicated media and ATP content was estimated by the firefly luciferase assay (27). The average nanomole of ATP per Klett unit of each culture is expressed as a percentage of the ATP content of cells in complete medium. TABLE 4. Levels ofppgpp in E. coli A-33 rela + and A-33 rela during starvation for inorganic ions' ppgpp (pmol/od) Medium A-33 rela + A-33 rela Complete 56 ± 19 (21) 52 ± 20 (20) -Arg,-trp 432 ± 37 (3) 40 ± 6 (3) - K+ 321 ± 179 (8) 73 ± 30 (15) - Mg ± 65 (16) 111 ± 71 (15) a Growing cells were collected on a membrane filter (Millipore Corp.) and washed four times with lowphosphate Tris medium lacking one of the indicated nutrients. Cells were suspended in the same medium or in complete medium. A portion of each culture was supplemented with 100, Ci of '2P, per ml. At various intervals the levels of ppgpp were determined (1). Each value for ppgpp represents the mean ± standard deviation of the number of determinations indicated in the parentheses.

7 VOL. 143, 1980 of pyrophosphate from ATP to GTP to synthesize ppgpp (2). These inhibitory effects of chloramphenicol and tetracycline on protein breakdown possibly reflect either a requirement for the synthesis of a new protease or a regulatory protein or could result from some additional action of these inhibitors, such as might result from their binding to ribosomes. In fact, upon amino acid deprivation, tetracycline or chloramphenicol has been shown to reduce proteolysis by preventing accumulation ofppgpp, and not by their inhibition of protein synthesis (28). To examine this issue further, we studied the effect of ion starvation on proteolysis when protein synthesis was blocked by a mechanism that did not involve antibiotics, i.e., by using temperature-sensitive valyl-trna synthetase mutants. At the permissive temperature, 300C, both z I-~~~~~~~~~~o 0 IL 3 to.~ ~ ~ MINUTES P~~~~~~~~~r P- -P4 -Po +TET +T~~~~; ET FIG. 4. Effects of tetracycline on protein breakdown in E. coli A-33 rela + starved for phosphate or potassium ions. Protein breakdown was measured in complete medium (A) or in medium lacking phosphate (0) or potassium (E). At the time indicated by the arrow, each culture was divided in half, and tetracycline (50 pg/ml) was added to one portion: (A) complete medium + tetracycline; (0) phosphatefree + tetracycline; (5) potassium-free + tetracycline. PROTEIN BREAKDOWN IN E. COLI 1229 Effect of chloramphenicol on protein TABLE 5. degradation in cells starved for ionsa Protein breakdown Strain Culture medium (%/h) -CM + CM A-33 rela + Complete Potassium Phosphate Magnesium Nitrogen A-33 rela Complete 2.1 _b - Potassium Phosphate a Protein breakdown was measured as described in Table 1 in the presence or absence of chloramphenicol (CM) (100 ug/ml). Similar results were obtained in three separate experiments. b_, Not done. strains NF536 (rela +) and NF537 (rela) increased protein breakdown upon deprivation for inorganic ions (Table 6). At 39 C the valyltrna synthetase in these strains was completely inactivated and, in complete medium, there was a four- to fivefold increase in proteolysis in mutant NF536, but not in its relaxed counterpart (Table 6), in accord with earlier findings (28). The removal of inorganic ions from the medium had little, if any, effect on the accelerated protein catabolism in strain NF536, but led to a consistent increase in protein catabolism in strain NF537 (rela). Therefore, the stimulation in protein breakdown upon ion starvation does not require concomitant protein synthesis, and the inhibitory effects of chloramphenicol and tetracycline (Table 5, Fig. 4) must involve some additional mechanism. DISCUSSION These studies demonstrate that starvation for various inorganic nutrients not only causes inhibition of growth and RNA synthesis, but also leads to an increase in overall protein degradation (Table 1. See Table 7 for summary). For example, deprivation for potassium, like starvation for amino acids or a carbon source (11), stimulates the hydrolysis of the more stable cell proteins two- to threefold, whereas the breakdown of rapidly degraded normal (Fig. 2) or abnormal (Fig. 3) proteins is unaffected. It is also noteworthy that simultaneous starvation for a carbon or amino acid source and for inorganic ions does not have additive effects in promoting overall proteolysis. The changes in protein breakdown during various types of starvation are summarized in Table 7.

8 1230 ST. JOHN AND GOLDBERG Our previous studies demonstrated that rates of protein catabolism correlate with the intracellular levels of ppgpp (28, 29, 31) during various conditions. Thus, an increase in the overall rate of protein breakdown can occur by either of two mechanisms: (i) in rea+ cells deprived of amino acids (26, 30, 31) or amino acyl-trna (9, 28), synthesis of ppgpp increases, which in turn causes a stimulation of protein catabolism; (ii) TABLE 6. Effect of deprivation for valyl-trna on protein degradation in E. coli NF536 and NF537 during phosphate or magnesium starvation' % Protein Expt Strain Medium breakdown/2 h 30 C 390C 1 NF536 Complete Phosphate NF536 Complete Magnesium NF537 Complete Phosphate NF537 Complete Magnesium aat 39 C, growth of these strains stops as a consequence of inactivation of valyl-trna synthetase. Cells grown at 30 C were labeled for two generations with [3H]phenylalanine (0.1,uCi/ml). Cells were harvested and washed four times with starvation media supplemented with 120 jg of unlabeled phenylalanine per ml. Cells were suspended in complete medium or in media lacking phosphate or magnesium which had been prewarmed to the indicated temperatures. Protein breakdown was determined from the release of [3H]phenylalanine into an acid-soluble form. Similar results were obtained in two separate experiments. TABLE 7. J. BACTERIOL. during starvation of relx+ strains for an energy source or during any moderate reduction in ATP, a relu-independent accumulation of ppgpp (29, 30, 31) occurs, and as a result of reduced degradation of this nucleotide (3, 14, 29), leads to a stimulation of proteolysis. Furthernore, a variety of observations indicate that these correlations are not fortuitous (31) and that ppgpp stimulates this process (although we cannot eliminate the possibility that some metabolite of ppgpp [e.g., ppgp, 23] might not be the actual regulator of proteolysis). For example, protein catabolism falls to basal levels when ppgpp synthesis is inhibited (28,29,31), and the kinetics and concentration dependence of these changes in ppgpp and protein catabolism (31) all strongly support an essential role of ppgpp in signaling this adaptation to glucose and amino acid starvation (Table 4). Since the removal of potassium, phosphate, or magnesium from the growth media also leads to a moderate (40 to 70%) decrease in the intracellular ATP levels in E. coli (Table 3), we earlier suggested (11) that protein catabolism in such cells may increase by a mechanism similar to that seen during carbon starvation, i.e., that the fall in ATP in the ion-starved cells might lead to a decrease in ppgpp degradation which would result in an accumulation of this nucleotide. However, several findings argue strongly against such a mechanism. First of all, relx cells which do not accumulate ppgpp in response to carbon starvation (22) still increase protein breakdown normally during ion deprivation (Table 2). Furthernore, in the rela + and rela cells starved for various ions, the rates of protein catabolism do not correlate with the levels of ppgpp (Table 4). For example, no increase in ppgpp was found in Summary: conditions affecting overall protein degradation and ppgpp levels in various E. coli strains' rela+ relx+ rela reix+ rela reix Conditions Protein G Protein G Protein degrada- content degrada- content degrada- content tion tion tion ppgpp-dependent responsesb Cells deprived of: Amino acids Carbon source ppgpp-independent responses Cells deprived of: K ND Mg2e ND p ND + ND + ND athese results summarize the present findings as well as related results (28, 29, 31). Symbols: +, signifies an increase over basal levels; -, indicates no change; ND, not determined. b The conclusion for ppgpp involvement is based also on kinetic studies of ppgpp levels and protein degradation (31).

9 VOL. 143, 1980 potassium- or magnesium-starved reu cells which have an accelerated rate of protein breakdown. The exact reason why a fall in energy production did not lead to an accumulation of ppgpp under these conditions is not clear. Presumably the effects of ion starvation on energy metabolism involve very different mechanisms from those occurring upon glucose limitation or during treatment with inhibitors of respiration (29, 31). It is interesting in this context that ppgpp did accumulate in rela + cells deprived of potassium; possibly under this condition there is some alteration in the levels ofamino acyl-trna within the cells. In any case, these various observations clearly indicate that overall protein catabolism can increase during ion starvation by a ppgpp-independent mechanism (Table 7). In related studies (Rosenthal, Voellmy, and Goldberg, unpublished observations), protein degradation was also found to increase upon treatment with inhibitors of RNA synthesis, without any change in the intracellular level of ppgpp. It thus appears likely that the acceleration of protein degradation upon amino acid or glucose starvation and that occurring during ion deprivation involve distinct mechanisms. In addition, these different forms of starvation seem to affect the degradation of different classes of cell proteins even though the combined starvation for organic and inorganic nutrient does not cause additive increases in proteolysis. Related studies by Voellmy and Goldberg (manuscript in preparation) indicate that the lack of phosphate and magnesium ions leads to increased breakdown of ribosomal proteins, which as a class are stable in glucose- or amino acid-deprived cultures. The acceleration of overall proteolysis during deprivation for inorganic ions and that resulting from ppgpp accumulation probably represent very distinct biological responses having very different physiological significance. Inhibitors of protein synthesis have been shown to inhibit the increase in protein degradation in bacterial and animal cells during poor nutritional conditions. Hershko and colleagues (25, 26) and others (17, 30) have suggested that such inhibitors prevent the production of a labile polypeptide that is required for the stimulation of protein catabolism. In E. coli, such a hypothetical polypeptide must turn over rapidly since the basal rate of protein catabolism is reestablished within 15 to 20 min (29, 30) (Fig. 4) or much faster (31) after administration of inhibitors of protein synthesis. Various experiments, however, indicate that the reduction in proteolysis induced by chloramphenicol or tetracycline in cells starved for amino acid or an energy source results from the dramatic fall of ppgpp PROTEIN BREAKDOWN IN E. COLI 1231 content induced by these agents, rather than from the inhibition of protein synthesis per se (28-30). During starvation for phosphate, potassium, or magnesium ions, treatment with these inhibitors also causes a fall in the rate of proteolysis (Table 5). Since the stimulation of protein breakdown under such conditions seems to occur by a ppgpp-independent pathway, the effect of these antibiotics cannot be explained by a reduction in the ppgpp pool. When we utilized another approach to block protein synthesis (i.e., inactivation of a temperature-sensitive valyltrna-synthetase), we found that ion starvation could still induce an increase in the rate of protein catabolism. Thus, the acceleration of proteolysis under these conditions does not require concomitant protein synthesis, and the mechanism by which tetracycline and chloramphenicol influence protein degradation in ionstarved cells remains unclear. Understanding these anomalous effects of chloramphenicol and tetracycline should help clarify the mechanisms by which deprivation for phosphate, potassium, or magnesium leads to an acceleration of proteolysis. Magnesium, potassium, and phosphate are important for the stability of the ribosome. Under conditions in which such ions are lacking, the ribosome constitutes a large intracellular reservoir for these ions (8, 35). It has been shown by several groups that starvation for phosphate (18), magnesium (19, 20), or potassium (5) leads to the selective hydrolysis of ribosomal RNA. The breakdown of the RNA moiety of ribosomes should lead to the release of ribosomal proteins. Voellmy and Goldberg (manuscript in preparation) have, in fact, demonstrated rapid breakdown of ribosomal protein in cells starved for magnesium and phosphate. It has been shown that failure of a normal polypeptide to associate in its normal multimeric structure can lead to the recognition of the polypeptide as an abnormal protein and the selective hydrolysis of the unassociated subunit (11). A similar situation might occur if the 54 ribosomal proteins were released from the ribosome. In fact, in yeast (13), HeLa cells (33), and E. coli (4), the failure to produce ribosomal RNA or the excessive production of certain ribosomal proteins (6, 21) leads to a rapid degradation of the newly synthesized ribosomal proteins, which are unable to associate with RNA. Thus, the effect of the inhibitors of protein synthesis on protein degradation in the ion-starved cells may be the result of their ability to interact with the ribosome and to stabilize it. In fact, chloramphenicol is known to increase polysome stability during certain types of starvation (32). Presumably, any antibiotic that binds to ribosomes could serve to

10 1232 ST. JOHN AND GOLDBERG stabilize these structures and to reduce the release and subsequent hydrolysis of ribosomal proteins. Experiments to test this model are now in progress. According to this model, protein breakdown during ion starvation does not increase by a specific activation of the degradative machinery (as appears to occur when levels of ppgpp rise [28, 29, 31]), but instead increases in response to an alteration in the susceptibility of certain cell proteins to proteolysis. The lack of the appropriate ionic milieu under these conditions leads to a destabilization of certain proteins and to their selective hydrolysis (e.g., those in ribosomes and possibly others). The increased protein breakdown under these conditions thus serves a very different physiological purpose than that seen upon starvation for amino acids or glucose. Under the latter conditions, increasing proteolysis appears to be of selective advantage to the bacteria, since it provides a source of amino acids for new enzyme synthesis or for energy metabolism. This explanation cannot apply to ion starvation. In fact, supplying all amino acids in the medium does not reduce the rapid proteolysis seen in potassium-deprived cells, nor can amino acids relieve this nutritional deficiency. The increased proteolysis under these conditions would appear to protect the cell against the accumulation of an aberrant, and potentially harmful, class of intracellular proteins (10-12, 24) that are unable to function in the altered ionic milieu. ACKNOWLEDGMENTS These studies have been made possible by research grants to Ann St. John from the National Science Foundation (PCM ) and the Rutgers Research Council and to Alfred L. Goldberg from the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS). 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