Starvation Potassium Other Inorganic Ions Degradation … · NF859metB argA reUA+ reIX+,...

JOURNAL OF BACTERIOLOGY, Sept. 1980, p. 1223-12330021-9193/80/09-1223/11$02.00/0

Vol. 143, No. 3

Effects of Starvation for Potassium and Other Inorganic Ionson Protein Degradation and Ribonucleic Acid Synthesis in

Escherichia coliANN C. ST. JOHN'* AND ALFRED L. GOLDBERG2

Department ofMicrobiology, Bureau of Biological Research, Rutgers University, New Brunswick, NewJersey 08903,' and Physiology Department, Harvard Medical School, Boston, Massachusetts 021152

Starvation of Escherichia coli for potassium, phosphate, or magnesium ionsleads to a reversible increase in the rate of protein degradation and an inhibitionofribonucleic acid (RNA) synthesis. In cells deprived of potassium, the breakdownof the more stable cell proteins increased two- to threefold, whereas the hydrolysisof short-lived proteins, both normal ones and analog-containing polypeptides, didnot change. The mechanisms initiating the enhancement of proteolysis duringstarvation for these ions were examined. Upon starvation for amino acids oramino acyl-transfer RNA (tRNA), protein breakdown increases in reA+ (but notreA) 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 tobe responsible for the increased protein breakdown in cells starved for inorganicions, 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 thisresponse. In bacteria in which energy production is restricted, ppGpp levels alsorise, and protein breakdown increases. The ion-deprived cultures did show a 40 to75% reduction in adenosine-5'-triphosphate levels, similar to that seen uponglucose starvation. However, this decrease in ATP content does not appear tocause the increase in protein breakdown or lead to an accumulation of ppGpp. Noconsistent change in intracellular ppGpp levels was found in reA + or reA cellsstarved for these ions. In addition, in reiX mutants, removal of these ions led toaccelerated protein degradation even though relX cells are unable to increaseppGpp levels or proteolysis when deprived of' a carbon source. In the potas-sium-, phosphate-, and magnesium-deprived cultures, the addition of choram-phenicol or tetracycline caused a reduction in protein breakdown toward basallevels. Such findings, however, do not indicate that protein synthesis is essentialfor the enhancement of protein degradation, since blockage of protein synthesisby inactivation of a temperature-sensitive valyl-tRNA synthetase did not restoreprotein catabolism to basal levels. These various results and related studiessuggest that the mechanism for increased protein catabolism on starvation forinorganic ions differs from that occurring upon amino acid or carbon deprivationand probably involves an enhanced susceptibility of various cell proteins (espe-cially ribosomal proteins) to proteolysis.

In growing bacteria the average rates of pro-tein breakdown are relatively low, but whensuch cells are deprived of a carbon source, nitro-gen, or an essential amino acid, the degradationof cell proteins increases severalfold (11, 17, 24).It appears likely that the stimulation of prote-olysis is of value to the starving cell by providinga source of amino acids for further protein syn-thesis or for energy metabolism (11, 12). A largevariety of evidence indicates that this responserequires an accumulation of guanosine-5'-di-phosphate-3'-diphosphate (ppGpp) or someclosely related compound in the cells. During

starvation for an amino acid (11, 24, 26, 30) oramino acyl-tRNA (9, 28) protein degradationincreases in reA+ cells, but not relA mutants,which are unable to accumulate ppGpp undersuch conditions (2, 28, 31). Furthermore, theactual increase in protein catabolism under theseconditions is proportional to the increase inppGpp levels (31). In addition, when cells arestarved for a carbon source (29, 30, 31), or whenproduction of ATP is restricted (29), proteincatabolism increases in both reL4+ and relAstrains. Treatment of amino acid-deprived orenergy-depleted cells with agents that block

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1224 ST. JOHN AND GOLDBERG

ppGpp synthesis, such as tetracycline, leads toa rapid return of the rate of proteolysis to basallevels (29, 31). Finally, the rate of fall in prote-olysis under these conditions depends upon therate of disappearance of ppGpp (31).

Protein catabolism increases not only in re-sponse to starvation for carbon or nitrogensources, but also when cells are starved for in-organic nutrients, such as phosphate (26, 34) ormagnesium (34). The physiological significanceof this cellular response is not clear, since en-hanced proteolysis should not relieve the under-lying nutrient deficiency in the ion-starved cul-tures. It is also not known whether the lack ofinorganic ions affects the degradation of thesame proteins as does glucose or amino aciddeprivation.To learn more about the mechanisms regulat-

ing protein breakdown in E. coli, we have sys-tematically studied the response to starvationfor various essential ions. Protein catabolismwas found to increase not only during depriva-tion for phosphate and magnesium but alsowhen cultures lack potassium (11). Since star-vation for all these inorganic nutrients resultedin a similar increase in protein breakdown anda simultaneous decrease in RNA synthesis, acommon cellular mechanism may be responsiblefor these effects. For example, it was suggested(11) that the coordinate changes in proteolysisand RNA synthesis may be signaled by mecha-nisms similar to those occurring upon energystepdown, i.e., by a fall in cellular ATP levelsand a subsequent increase in ppGpp. The pres-ent studies examine more closely the regulationof protein catabolism during ion starvation andthe role of ATP levels and guanosine-bis-di-phosphate in this process.

MATERLU1S AND METHODSBacterial strains and media. Escherichia coli

strains A-33 reUA+ arg trpA and A-33 relA arg trpAwere kindly provided by B. Davis. E. coli strainsNF859 metB argA reUA+ reIX+, NF859X metB reArelX+, and NF1035 metB relA relX were kindly pro-vided by J. Gallant. E coli mutants NF536 leuvalS(Ts) reU+ and NF537 leu valS(Ts) relA werekindly provided by J. D. Friesen.

Cells were grown with aeration at 37°C in either thebasal salts medium described previously (9) or in Trismedium (pH 7.4) containing (per liter): 7.26 g of Tris,2.4 g of (NH4)2SO4, 0.295 g of MgSO4. 7H20, 0.298 g ofKCI, 0.268 g of Na2HPO4.7H20, and 0.174 g of NaCI.Glucose (5 g/liter) and required amino acids (30 mg/liter) were sterilized separately. To prepare starvationmedia, the following changes in the composition ofTris medium were made: for potassium-free, KCI wasomitted; for magnesium-free, MgSO4 7H20 was omit-ted; 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 measur-ing turbidity of the cell suspension with a Klett-Sum-merson colorimeter (green filter) or a Gilford spectro-photometer at 550 nm. An optical density at 550 nm(OD5r,m) of 1.0 was equivalent to 95 Klett units and 4x 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 grownfor two generations in Tris medium containing L-4,5-[3H]leucine (0.1 uCi/ml). The cells were collected onMillipore filters and washed four times with starvationmedium containing 120 Lg of unlabeled leucine per ml.The cells were suspended at a density of 2 x 108 to 3x 10' cells/ml in medium containing 120,ug of unla-beled leucine per ml. At various times, 1.0-ml sampleswere removed and added to 0.1 ml of 50% trichloro-acetic acid. Degradation of protein was measured fromthe release of [3H]leucine into acid-soluble form asdescribed previously (9). Each value is the average oftwo determinations which generally agreed within 5%.In strains NF536 and NF537, the breakdown of pro-teins labeled with ['Hlphenylalanine (0.2 PCi/ml) wasmeasured as described previously (28). To measurethe breakdown of amino acid analog-containing pro-tein, strain A-33 relA + cells were washed two times inarginine-free medium containing 2.1 ,uCi of ['IC]-canavanine per ml. After 19 min, the cells were washedin ion-free medium and suspended at a density of 3 x108 cells/ml in medium containing 120 ug of arginineper ml. The release of ['4C]canavanine into trichloro-acetic acid-soluble form was measured as describedpreviously (28).Measurement of net RNA synthesis. RNA

synthesis was measured by the incorporation of 2-['4C]uracil into material precipitable with 5% trichlo-roacetic acid which could be hydrolyzed by treatmentwith 1.0 N KOH for 16 h at 370C as described previ-ously (29). The growth medium was supplementedwith ['4C]uracil (0.05 ,uCi/ml, specific activity 0.56 uCi/,umol). RNA content was also determined colorimet-rically with the orcinol reaction (16).Measurement of nucleotides. The levels of ATP

were measured after extracting the cells with 5% tri-chloroacetic acid. The acid was extracted with fourvolumes of ether, and ATP was measured in duplicateby the firefly luciferase assay as described by Stanleyand Williams (27). Lyophilized firefly lanterns wereobtained from Sigma Chemical Co (St. Louis, Mo.).To measure ppGpp levels, cells were collected on

Millipore filters and washed four times with the low-phosphate Tris medium previously described (29)lacking one of the essential nutrients as described inthe experimental results. Cells were resuspended inthe same medium at a density of 1 x 108 cells/ml. Aportion of the culture was used to determine cellturbidity (ODm%), and another portion was supple-mented with 100 ,uCi of 32P per ml. Levels of ppGppwere estimated by the method of Cashel (1).

RESULTSEffect of starvation for potassium on pro-

tein degradation and RNA synthesis inreIA+ cells. During starvation for a source of


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carbon, nitrogen, or amino acids, E. coli cellsincrease their degradation of preexistent pro-teins, apparently to provide a source of aminoacids for protein synthesis. These cells also de-crease the synthesis of rRNA and tRNA. Theeffects of potassium starvation on protein break-down and RNA synthesis were therefore exam-ined in E. coli A-33 reMA. In a typical experi-ment, growing cells were transferred to mediumlacking potassium ions, at which point growthslowed and then stopped within 60 min. Therate of breakdown of preexistent proteins in-creased 2- to 2.5-fold upon suspension of thecells in potassium-free medium (Fig. 1). Whenpotassium was resupplied to the starved cells,growth resumed and protein breakdown re-turned to basal levels (Fig. 1). The increase inprotein catabolism in potassium-deprived celLsprobably did not result from a secondary re-quirement for potassium for the biosynthesis ofcertain amino acids or the transport of otherions, such as phosphate. Accordingly, the addi-tion of either a complete mixture of amino acidsor a 250-fold increase in the molarity of phos-phate did not reduce protein degradation orpermit growth to resume. During potassiumstarvation, RNA synthesis was greatly reducedas measured by the incorporation of radioactiveuracil (Table 1). Such changes do not reflectalterations in uracil uptake since similar de-creases were seen when RNA accumulation wasmeasured colorimetrically.The regulation of protein degradation during

starvation for carbon, nitrogen or amino acids

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FIG. 1. Effect ofpotassium deprivation on the rateof protein breakdown in strain A-33 reUA +. Proteinbreakdown was measured in complete medium (O) orin medium lacking potassium (-). At the time indi-cated by the arrow, 4 mM KCl was added to theculture in potassium-free medium (0).

PROTEIN BREAKDOWN IN E. COLI 1225

TABLE 1. Effect of starvation on protein breakdownand net RNA synthesis in E. coli A-33 relA + and A-

33 reLAa

A-33 relA+ A-33 relA

Uracil UracilMedium Protein incor- Protein incor-break- pora- break- pora-

down tion (% down tion (%(%/h) con- (%/h) con-

trol) trol)Complete 2.0 100 1.7 100-Arg, - trp 3.1 3 1.8 109- N, - arg, 5.0 1 4.0 7- trp

- Glucose 4.7 2 3.8 1- K+ 4.8 12 3.0 6- P043- 5.5 2 4.4 3-Mg2+ 4.8 20 3.8 24'To measure protein breakdown, cells were grown

for two generations in complete medium containing['Hjleucine (0.1 AtCi/ml). Cells were collected on mem-brane filters (Millipore Corp., Bedford, Mass.) andwashed four times with starvation medium containing120 ug of unlabeled leucine per ml. The labeled cellswere suspended in complete medium or in media lack-ing the indicated nutrient(s). Protein breakdown wasdetermined as described in the text. Similar resultswere obtained in four separate experiments. To mea-sure net RNA synthesis, growing cells were collectedon membrane filters (Millipore Corp.) and washedfour times with starvation medium containing 10 jig ofuracil per ml. The cells were suspended in completemedium or media lacking the indicated nutrient(s)supplemented with [14C]uracil (0.05 ,uCi/ml, specificactivity 0.56 MCi/,umol). Incorporation of ['4C]uracilwas determined as described in the text and expressedas a percentage of the incorporation found in completemedium. Similar results were found in two independ-ent experiments.

involves a preferential stimulation of the degra-dation of more stable polypeptides (11, 24). Nor-mal proteins that have short half-lives or pro-teins with abnormal structures are degraded ata rapid rate in the presence or absence of re-quired nutrients. We examined whether thestimulation of protein catabolism in potassium-starved cultures also involved a selective degra-dation of normally stable components. Becausethe half-lives of cellular proteins are heteroge-neous, the exposure of cells to radioactive aminoacids for various periods of time will preferen-tially label groups of proteins having differentstabilities (11). E. coli A-33 reLA+ were exposedto [3H]leucine for a short period (5 min) toenrich for radioactivity in the more labile frac-tion of proteins. The culture was divided, andprotein breakdown was immediately measuredin one-half of the culture in the presence andabsence of potassium (Fig. 2A). During the sub-


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FIG. 2. Effects ofpotassium deprivation on the degradation of the more labile recently synthesizedproteinfraction and the more stable protein fraction in E. coli A-33 reLA +. Cells at a density of5 x 108 cells/ml wereexposed to 0.2 ,uCi of[3HJleucine per ml for 5 min. (A) A portion of the culture was washed with potassium-free medium and suspended in complete medium (0) or in medium lacking potassium (0) to measuredegradation of the more rapidly degraded fraction of cell proteins; (B) a second portion of the culture waswashed and suspended in complete medium containing 120mM unlabeled leucine. After 60 min, the cells werewashed with potassium-free medium and suspended in complete medium (0) or medium lacking potassium(0) to measure the degradation ofmore stable proteins.

sequent 60 min, the degradation of labile com-ponents was primarily responsible for the ob-served rate of catabolism, and the rates of pro-tein breakdown were similar in the presence andabsence of potassium. The second half of thelabeled culture was allowed to grow in completemedium containing excess unlabeled leucine for60 min to allow for the breakdown of labilecomponents. After this treatment, proteinbreakdown of this culture was measured in thepresence and absence of potassium (Fig. 2B). Athreefold increase in the catabolism of labeledproteins was seen in the cells deprived of potas-sium. To examine the effect of potassium star-vation on the degradation of abnormal proteins,E. coli A-33 reA+ were labeled in medium inwhich arginine was replaced by the arginineanalog ['4C]canavanine. After transfer to me-dium containing arginine, the breakdown of theproteins containing ["Cicanavanine was fol-lowed in the presence and absence of potassium.As shown in Fig. 3, the rates of catabolism ofthese abnormal proteins were very similar inboth cultures. Therefore, like other types ofstarvation, potassium deprivation selectivelystimulates the breakdown of the more stable cellproteins.Starvation for other inorganic nutrients.

PROTEINS CONTAINING 14C-CANAVANINE

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FIG. 3. Effect of potassium deprivation on thebreakdown of abnormal proteins in E. coli A-33reA . Degradation of [''Cicanavanine-containingproteins was measured in complete medium (0) andin medium lacking potassium (0).

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Phosphate or magnesium starvation also causeda two- to threefold stimulation in protein deg-radation (Table 1). The rate of proteolysis underthese conditions was similar to that found incells starved for glucose or nitrogen. This en-hanced rate of proteolysis may be the maximalpossible rate of protein catabolism since starva-tion for more than one required nutrient did notlead to additive increases in proteolysis: proteindegradation was 5% per h in nitrogen and aminoacid-free medium, 4.8% per h in potassium-freemedium, and 5.5% per h in medium lacking allthree nutrients. Starvation for phosphate andmagnesium also led to a marked decrease inRNA synthesis, as had previously been reported(18, 26).Effect of starvation for inorganic nutri-

ents in relaxed cells. Cells that have a defectin the stringent control system (relA) are unableto increase protein degradation or reduce RNAsynthesis in response to starvation for aminoacids or amino acyl-tRNA (9, 28, 30) (Table 1).However, both these strains show the normalincrease in protein breakdown and decrease inRNA synthesis when deprived of a carbonsource (29, 30) (Table 1) or when energy pro-duction is reduced (29). Likewise, in E. coli A-33reA, the rate of protein catabolism increasedtwofold and RNA synthesis dropped 10-fold inthe absence of a nitrogen source. DecreasedRNA synthesis upon nitrogen starvation hasbeen seen in other relA strains (15). It should beemphasized that nitrogen deprivation is notidentical to amino acid starvation. In cells lack-ing a nitrogen source, the availability of nitrog-enous bases also decreases and, in related stud-ies, purine or pyrimidine deprivation has beenshown to accelerate proteolysis in relA strainsby a mechanism that appears to be independentof levels of ppGpp (Goldberg and Rosenthal,unpublished data). The effect of starvation forinorganic nutrients was examined in E. coli A-33 reA to determine whether the regulation ofproteolysis resembles that during starvation fora carbon source or starvation for amino acids.Protein catabolism was stimulated approxi-mately twofold by the removal of glucose, nitro-gen, potassium, phosphate, or magnesium (Ta-ble 1). Conditions in which catabolism was in-creased also led to a large decrease in the rate ofRNA synthesis.Protein degradation in reiX cells. Since

protein degradation and RNA synthesis appearto change coordinately during a number of star-vation conditions (11; Table 1), it is attractive tosuggest that a common mechanism regulatesboth processes. Although there is an excellentcorrelation between the levels of ppGpp and the

rates ofRNA synthesis (2, 7) and protein break-down (28) when amino acyl-tRNA is limiting,the kinetics of ppGpp accumulation do not cor-relate completely with rates of RNA synthesisduring carbon starvation and certain other con-ditions (2, 14). To test for a possible role ofppGpp in regulating proteolysis during starva-tion for glucose or inorganic nutrients, we ex-amined a mutant (relA relX) recently describedby Pao and Gallant (22). This strain has a verylow basal level of ppGpp and is unable to accu-mulate this nucleotide during starvation for glu-cose as a result of the reiX mutation. The ratesof protein degradation were measured in theisogenic series, NF859 (reA+ reLK+), NF859X(reU relX+), and NF1035 (relA reiX) duringincubation in complete medium or in mediaeither lacking glucose or the required amino acidmethionine (Table 2). As expected, starvationfor methionine caused a twofold stimulation ofproteolysis in E. coli NF859 but not in the reUstrains, which do not accumulate ppGpp in theabsence of required amino acids. During glucosestarvation the relX+ strains showed a two- tothreefold enhancement in the rate of proteindegradation, whereas mutant NF1035 showedno such stimulation of protein breakdown inaccord with earlier data (31). To examinewhether the regulation of protein catabolismduring starvation for inorganic ions was de-pendent on the reiX gene product, strain NF859,NF859X, or NF1035 was placed in medium lack-ing potassium, phosphate, or magnesium ions.All three strains were able to increase proteincatabolism severalfold (Table 2). Thus, the stim-ulation of protein catabolism during ion starva-tion occurs normally in the absence offunctionalrelA and relX gene products and differs fromthat seen in energy-restricted cells.Levels of ATP and ppGpp in ion-starved

cells. The coordinate changes in protein catab-olism and RNA synthesis during potassium,magnesium, or phosphate starvation of E. coliA-33 reLA + and A-33 relA cells (Table 1) suggestthat a common metabolic signal may regulatethese processes. It has previously been shownthat a moderate inhibition (30 to 50%) of thecell's ability to generate ATP leads to a stimu-lation of protein catabolism as well as a reduc-tion in growth and RNA synthesis in both reU +and reA strains. Therefore, the effect of star-vation for inorganic ions on cellular ATP levelswas examined (Table 3). Starvation for ions orfor glucose led to a 40 to 75% decrease in ATPcontent of the cells. Deprivation for a source ofpotassium or magnesium led to a decrease inATP similar to that induced by concentrationsof respiratory inhibitors (KCN or NaNA), that

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TABLE 2. Effect of various types of starvation onprotein breakdown in relA and reA reLY strainsa

Protein breakdown(%/2 h)

Experi- Meiument Mulum NF859 NF859X NF1035

(reLA+ (relA (relAreLX+) reLY') relX)

1 Complete 1.1 1.7 1.4- Glucose 3.7 3.8 1.5- Methio- 2.1 1.8 1.8

nine

2 Complete 2.0 2.1 1.6- Mg2+ 3.9 3.8 2.9

3 Complete 0.6 0.5 0.8- P043- 4.7 6.1 4.5

4 Complete 1.9 1.0 0.9-K+ 4.2 2.1 1.8

a Rates of protein degradation were measured in theusual fashion. The indicated strains were grown in thepresence of [3H]leucine for two generations to labelcell proteins. The cells were harvested and washedfour times with starvation medium supplemented with120 ,ug of unlabeled leucine per ml. The celLs weresuspended in a complete medium or in medium lackingeither glucose, methionine (a required amino acid),phosphate, or potassium. Similar results were obtainedin two separate experiments.

caused a twofold increase in protein catabolism.Phosphate starvation led to a 75% decrease inATP, which was similar to the reduction foundin glucose-starved cells.During carbon starvation or inhibition of en-

ergy production, an accumulation of ppGpp oc-curs in both rel + and relA cells as a result ofa reduced rate of degradation of this nucleotide(2, 3, 28). We therefore examined whether theenhancement of protein degradation in the ion-starved cells was the result of an increase inppGpp levels that might result from the fall incellular ATP content. The ppGpp levels of E.coli A-33 reA+ and A-33 rel were measuredduring starvation for potassium and magnesiumions (Table 4). Upon starvation for amino acids,the levels ofppGpp increased seven- to eightfoldin the rel + strain but did not change in therel cells. In potassium-starved E. coli A-33reUA , the ppGpp content was 2.5- to 9-foldgreater than in control cells. In the reA cells,however, no significant increase in ppGpp wasfound. When rel+ or rel cells were deprivedof magnesium, no consistent increase above thebasal ppGpp level was found, although therewere considerable variations in the levels of gua-nosine-bis-diphosphate. Thus, the stimulation ofprotein degradation in potassium-starved reAcells and magnesium-starved cells (unlike that

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seen on glucose or amino acid starvation) doesnot appear to require an accumulation ofppGpp.Effect of chloramphenicol and tetracy-

cline. The stimulation of protein catabolismduring glucose, nitrogen, or amino acid starva-tion can be reversed by the addition of theantibiotics chloramphenicol or tetracycline (11,17, 24). As shown in Fig. 4, the addition oftetracycline also lowered the rate of proteinbreakdown towards basal levels in culturesstarved for potassium or phosphate. Similar re-ductions in protein breakdown were found uponaddition of chloramphenicol to cells starved forinorganic ions or nitrogen (Table 5). These in-hibitors interact with the ribosome and therebyblock protein synthesis. Consequently, they in-duce a marked fall in the intracellular concen-tration of ppGpp because they indirectly permitintracellular pools of amino acid and RNA toincrease 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 reUAduring starvation for inorganic nutrientsa

ATPMedium (% control)

Complete ........................... 100- Glucose ........................ 25- K+. ............................. 60- P04 ........ .................. 27-m e. .......... 50a Cells were collected on membrane filters (Milli-

pore Corp.) and washed four times with the variousmedia. Cells were suspended in the indicated mediaand ATP content was estimated by the firefly lucifer-ase assay (27). The average nanomole of ATP perKlett unit of each culture is expressed as a percentageof the ATP content of cells in complete medium.

TABLE 4. Levels ofppGpp in E. coli A-33 relA + andA-33 relA during starvation for inorganic ions'

ppGpp (pmol/OD)Medium

A-33 relA + A-33 reLAComplete 56 ± 19 (21) 52 ± 20 (20)-Arg,-trp 432 ± 37 (3) 40 ± 6 (3)-K+ 321 ± 179 (8) 73 ± 30 (15)- Mg2+ 118 ± 65 (16) 111 ± 71 (15)a Growing cells were collected on a membrane filter

(Millipore Corp.) and washed four times with low-phosphate Tris medium lacking one of the indicatednutrients. Cells were suspended in the same mediumor in complete medium. A portion of each culture wassupplemented with 100, Ci of '2P, per ml. At variousintervals the levels of ppGpp were determined (1).Each value for ppGpp represents the mean ± standarddeviation of the number of determinations indicatedin the parentheses.


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of pyrophosphate from ATP to GTP to synthe-size ppGpp (2).These inhibitory effects of chloramphenicol

and tetracycline on protein breakdown possiblyreflect either a requirement for the synthesis ofa new protease or a regulatory protein or couldresult from some additional action of these in-hibitors, such as might result from their bindingto ribosomes. In fact, upon amino acid depriva-tion, tetracycline or chloramphenicol has beenshown to reduce proteolysis by preventing ac-cumulation ofppGpp, and not by their inhibitionof protein synthesis (28).To examine this issue further, we studied the

effect of ion starvation on proteolysis when pro-tein synthesis was blocked by a mechanism thatdid not involve antibiotics, i.e., by using temper-ature-sensitive valyl-tRNA synthetase mutants.At the permissive temperature, 300C, both

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FIG. 4. Effects of tetracycline on protein break-down in E. coli A-33 relA + starved for phosphate orpotassium ions. Protein breakdown was measured incomplete medium (A) or in medium lacking phos-phate (0) or potassium (E). At the time indicated bythe arrow, each culture was divided in half, andtetracycline (50 pg/ml) was added to one portion:(A) complete medium + tetracycline; (0) phosphate-free + tetracycline; (5) potassium-free + tetracycline.

TABLE 5. Effect of chloramphenicol on proteindegradation in cells starved for ionsa

Protein breakdownStrain Culture medium (%/h)

-CM +CM

A-33 relA + Complete 2.0 2.5- Potassium 4.8 2.7- Phosphate 5.5 3.2- Magnesium 4.8 2.8- Nitrogen 5.0 2.5

A-33 relA Complete 2.1 _b- Potassium 3.2 1.8- Phosphate 4.9 1.8

a Protein breakdown was measured as described inTable 1 in the presence or absence of chloramphenicol(CM) (100 ug/ml). Similar results were obtained inthree separate experiments.b_, Not done.

strains NF536 (relA+) and NF537 (reLA) in-creased protein breakdown upon deprivation forinorganic ions (Table 6). At 39°C the valyl-tRNA synthetase in these strains was com-pletely inactivated and, in complete medium,there was a four- to fivefold increase in proteol-ysis in mutant NF536, but not in its relaxedcounterpart (Table 6), in accord with earlierfindings (28). The removal of inorganic ions fromthe medium had little, if any, effect on theaccelerated protein catabolism in strain NF536,but led to a consistent increase in protein catab-olism in strain NF537 (reLA). Therefore, thestimulation in protein breakdown upon ion star-vation does not require concomitant protein syn-thesis, and the inhibitory effects of chloram-phenicol and tetracycline (Table 5, Fig. 4) mustinvolve some additional mechanism.

DISCUSSION

These studies demonstrate that starvation forvarious inorganic nutrients not only causes in-hibition of growth and RNA synthesis, but alsoleads to an increase in overall protein degrada-tion (Table 1. See Table 7 for summary). Forexample, deprivation for potassium, like starva-tion for amino acids or a carbon source (11),stimulates the hydrolysis of the more stable cellproteins two- to threefold, whereas the break-down of rapidly degraded normal (Fig. 2) orabnormal (Fig. 3) proteins is unaffected. It isalso noteworthy that simultaneous starvationfor a carbon or amino acid source and for inor-ganic ions does not have additive effects in pro-moting overall proteolysis. The changes in pro-tein breakdown during various types of starva-tion are summarized in Table 7.

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Our previous studies demonstrated that ratesof protein catabolism correlate with the intra-cellular levels of ppGpp (28, 29, 31) during var-ious conditions. Thus, an increase in the overallrate of protein breakdown can occur by either oftwo mechanisms: (i) in reA+ cells deprived ofamino acids (26, 30, 31) or amino acyl-tRNA (9,28), synthesis of ppGpp increases, which in turncauses a stimulation of protein catabolism; (ii)

TABLE 6. Effect of deprivation for valyl-tRNA onprotein degradation in E. coli NF536 and NF537

duringphosphate or magnesium starvation'% Protein

Expt Strain Medium breakdown/2 h30°C 390C

1 NF536 Complete 1.7 8.1- Phosphate 4.9 9.0

2 NF536 Complete 1.6 6.1- Magnesium 2.5 6.8

3 NF537 Complete 1.5 4.4- Phosphate 5.1 8.5

4 NF537 Complete 1.2 3.3- Magnesium 2.1 4.6

aAt 39°C, growth of these strains stops as a conse-quence of inactivation ofvalyl-tRNA synthetase. Cellsgrown at 30°C were labeled for two generations with[3H]phenylalanine (0.1 ,uCi/ml). Cells were harvestedand washed four times with starvation media supple-mented with 120 jg of unlabeled phenylalanine perml. Cells were suspended in complete medium or inmedia lacking phosphate or magnesium which hadbeen prewarmed to the indicated temperatures. Pro-tein breakdown was determined from the release of[3H]phenylalanine into an acid-soluble form. Similarresults were obtained in two separate experiments.

during starvation of relX+ strains for an energysource or during any moderate reduction inATP, a reLU-independent accumulation ofppGpp (29, 30, 31) occurs, and as a result ofreduced degradation of this nucleotide (3, 14,29), leads to a stimulation of proteolysis. Fur-thernore, a variety of observations indicate thatthese correlations are not fortuitous (31) andthat ppGpp stimulates this process (although wecannot eliminate the possibility that some me-tabolite of ppGpp [e.g., ppGp, 23] might not bethe actual regulator of proteolysis). For example,protein catabolism falls to basal levels whenppGpp synthesis is inhibited (28,29,31), and thekinetics and concentration dependence of thesechanges in ppGpp and protein catabolism (31)all strongly support an essential role of ppGppin signaling this adaptation to glucose and aminoacid starvation (Table 4).

Since the removal of potassium, phosphate, ormagnesium from the growth media also leads toa moderate (40 to 70%) decrease in the intracel-lular ATP levels in E. coli (Table 3), we earliersuggested (11) that protein catabolism in suchcells may increase by a mechanism similar tothat seen during carbon starvation, i.e., that thefall in ATP in the ion-starved cells might lead toa decrease in ppGpp degradation which wouldresult in an accumulation of this nucleotide.However, several findings argue strongly againstsuch a mechanism. First of all, relX cells whichdo not accumulate ppGpp in response to carbonstarvation (22) still increase protein breakdownnormally during ion deprivation (Table 2). Fur-thernore, in the relA + and relA cells starved forvarious ions, the rates of protein catabolism donot correlate with the levels ofppGpp (Table 4).For example, no increase in ppGpp was found in

TABLE 7. Summary: conditions affecting overall protein degradation andppGpp levels in various E. colistrains'

relA+ relX+ relA reIX+ relA reiX

Conditions Protein G Protein G Proteindegrada- content degrada- content degrada- content

tion tion tion

ppGpp-Dependent responsesbCells deprived of:Amino acids + + - - - -Carbon source + + + +

ppGpp-Independent responsesCells deprived of:K+ + + + - + NDMg2e + + - + NDp033- + ND + ND + ND

aThese results summarize the present findings as well as related results (28, 29, 31). Symbols: +, signifies anincrease over basal levels; -, indicates no change; ND, not determined.

bThe conclusion for ppGpp involvement is based also on kinetic studies of ppGpp levels and proteindegradation (31).

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potassium- or magnesium-starved reU cellswhich have an accelerated rate of protein break-down. The exact reason why a fall in energyproduction did not lead to an accumulation ofppGpp under these conditions is not clear. Pre-sumably the effects of ion starvation on energymetabolism involve very different mechanismsfrom those occurring upon glucose limitation orduring treatment with inhibitors of respiration(29, 31). It is interesting in this context thatppGpp did accumulate in relA + cells deprived ofpotassium; possibly under this condition there issome alteration in the levels ofamino acyl-tRNAwithin the cells. In any case, these various ob-servations clearly indicate that overall proteincatabolism can increase during ion starvation bya ppGpp-independent mechanism (Table 7). Inrelated studies (Rosenthal, Voellmy, and Gold-berg, unpublished observations), protein degra-dation was also found to increase upon treat-ment with inhibitors ofRNA synthesis, withoutany change in the intracellular level of ppGpp.

It thus appears likely that the acceleration ofprotein degradation upon amino acid or glucosestarvation and that occurring during ion depri-vation involve distinct mechanisms. In addition,these different forms of starvation seem to affectthe degradation of different classes of cell pro-teins even though the combined starvation fororganic and inorganic nutrient does not causeadditive increases in proteolysis. Related studiesby Voellmy and Goldberg (manuscript in prep-aration) indicate that the lack of phosphate andmagnesium ions leads to increased breakdownof ribosomal proteins, which as a class are stablein glucose- or amino acid-deprived cultures. Theacceleration of overall proteolysis during depri-vation for inorganic ions and that resulting fromppGpp accumulation probably represent verydistinct biological responses having very differ-ent physiological significance.

Inhibitors of protein synthesis have beenshown to inhibit the increase in protein degra-dation in bacterial and animal cells during poornutritional conditions. Hershko and colleagues(25, 26) and others (17, 30) have suggested thatsuch inhibitors prevent the production ofa labilepolypeptide that is required for the stimulationof protein catabolism. In E. coli, such a hypo-thetical polypeptide must turn over rapidly sincethe basal rate of protein catabolism is reestab-lished within 15 to 20 min (29, 30) (Fig. 4) ormuch faster (31) after administration of inhibi-tors of protein synthesis. Various experiments,however, indicate that the reduction in proteol-ysis induced by chloramphenicol or tetracyclinein cells starved for amino acid or an energysource results from the dramatic fall of ppGpp

content induced by these agents, rather thanfrom the inhibition of protein synthesis per se(28-30). During starvation for phosphate, potas-sium, or magnesium ions, treatment with theseinhibitors also causes a fall in the rate of prote-olysis (Table 5). Since the stimulation of proteinbreakdown under such conditions seems to occurby a ppGpp-independent pathway, the effect ofthese antibiotics cannot be explained by a re-duction in the ppGpp pool. When we utilizedanother approach to block protein synthesis (i.e.,inactivation of a temperature-sensitive valyl-tRNA-synthetase), we found that ion starvationcould still induce an increase in the rate ofprotein catabolism. Thus, the acceleration ofproteolysis under these conditions does not re-quire concomitant protein synthesis, and themechanism by which tetracycline and chloram-phenicol influence protein degradation in ion-starved cells remains unclear.Understanding these anomalous effects of

chloramphenicol and tetracycline should helpclarify the mechanisms by which deprivation forphosphate, potassium, or magnesium leads to anacceleration of proteolysis. Magnesium, potas-sium, and phosphate are important for the sta-bility ofthe ribosome. Under conditions in whichsuch ions are lacking, the ribosome constitutesa large intracellular reservoir for these ions (8,35). It has been shown by several groups thatstarvation for phosphate (18), magnesium (19,20), or potassium (5) leads to the selective hy-drolysis of ribosomal RNA. The breakdown ofthe RNA moiety of ribosomes should lead to therelease ofribosomal proteins. Voellmy and Gold-berg (manuscript in preparation) have, in fact,demonstrated rapid breakdown of ribosomalprotein in cells starved for magnesium and phos-phate. It has been shown that failure of a normalpolypeptide to associate in its normal multimericstructure can lead to the recognition of the poly-peptide as an abnormal protein and the selectivehydrolysis of the unassociated subunit (11). Asimilar situation might occur if the 54 ribosomalproteins were released from the ribosome. Infact, in yeast (13), HeLa cells (33), and E. coli(4), the failure to produce ribosomal RNA or theexcessive production of certain ribosomal pro-teins (6, 21) leads to a rapid degradation of thenewly synthesized ribosomal proteins, which areunable to associate with RNA. Thus, the effectof the inhibitors of protein synthesis on proteindegradation in the ion-starved cells may be theresult of their ability to interact with the ribo-some and to stabilize it. In fact, chloramphenicolis known to increase polysome stability duringcertain types of starvation (32). Presumably, anyantibiotic that binds to ribosomes could serve to

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stabilize these structures and to reduce therelease and subsequent hydrolysis of ribosomalproteins. Experiments to test this model are nowin progress.According to this model, protein breakdown

during ion starvation does not increase by aspecific activation of the degradative machinery(as appears to occur when levels of ppGpp rise[28, 29, 31]), but instead increases in response toan alteration in the susceptibility of certain cellproteins to proteolysis. The lack of the appro-priate ionic milieu under these conditions leadsto a destabilization of certain proteins and totheir selective hydrolysis (e.g., those in ribo-somes and possibly others). The increased pro-tein breakdown under these conditions thusserves a very different physiological purposethan that seen upon starvation for amino acidsor glucose. Under the latter conditions, increas-ing proteolysis appears to be of selective advan-tage to the bacteria, since it provides a source ofamino acids for new enzyme synthesis or forenergy metabolism. This explanation cannot ap-ply to ion starvation. In fact, supplying all aminoacids in the medium does not reduce the rapidproteolysis seen in potassium-deprived cells, norcan amino acids relieve this nutritional defi-ciency. The increased proteolysis under theseconditions would appear to protect the cellagainst the accumulation of an aberrant, andpotentially harmful, class of intracellular pro-teins (10-12, 24) that are unable to function inthe altered ionic milieu.

ACKNOWLEDGMENTSThese studies have been made possible by research grants

to Ann St. John from the National Science Foundation (PCM7682995) and the Rutgers Research Council and to Alfred L.Goldberg from the National Institute of Neurological andCommunicative Disorders and Stroke (NINCDS).

The authors are grateful to Eric Rosenthal, N. Fedele, andR. Misra for their expert technical assistance and to Elsa Foxand Maureen Rush for aiding us in the preparation of thismanuscript.

LITERATURE CITED1. Cashel, M. 1969. The control of ribonucleic acid synthesis

in Escherichia coli. IV. Relevance of unusual phospho-rylated compounds from amino acid starved stringentstrains. J. Biol. Chem. 244:3133-3141.

2. Cashel, M. 1975. Regulation of bacterial ppGpp andpppGpp. Annu. Rev. Microbiol. 29:301-318.

3. De Boer, H. A., A. J. Bakker, W. J. Weyer, and M.Gruber. 1976. The role of energy-generating processesin the degradation of guanosine tetraphosphate, ppGpp,in Escherichia coli. Biochim. Biophys. Acta 432:361-368.

4. Dennis, P. P. 1974. Synthesis and stability of individualribosomal proteins in the presence of rifampicin. Mol.Gen. Genet. 134:3947.

5. Ennis, H. L., and M. Lubin. 1975. Pre-ribosomal parti-cles formed in potassium-depleted cells: studies on deg-radation and stabilization. Biochim. Biophys. Acta 95:605-623.

6. Fallon, A. M1., C. S. Jinks, M. Yamamoto, and M.Nomura. 1979. Expression of ribosomal protein genescloned in a hybrid plasmid in Escherichia coli: genedosage effects on synthesis of ribosomal proteins andribosomal protein messenger ribonucleic acid. J. Bac-teriol. 138:383-396.

7. Fiil, N. P., K. von Meyenburg, and J. D. Friesen. 1972.Accumulation and turnover of guanosine tetraphos-phate in Escherichia coli. J. Mol. Biol. 71:769-783.

8. Goldberg, A. L. 1966. Magnesium binding by E. coliribosomes. J. Mol. Biol. 15:663-673.

9. Goldberg, A. L 1971. A role of amino acyl-tRNA in theregulation of protein breakdown in Escherichia coli.Proc. Natl. Acad. Sci. U.S.A. 68:362-366.

10. Goldberg, A. L., J. D. Kowit, J. D. Etlinger, and Y.Klemes. 1978. Selective degradation of abnormal pro-teins in animal and bacterial cells, p. 177-196. In D.Doyle and H. Segal (ed.), Protein turnover and lyso-somal function, SUNY Symposium, Academic Press,Inc., New York.

11. Goldberg, A. L., and A. C. St. John. 1976. Intracellularprotein degradation in mammalian and bacterial cells:part 2. Annu. Rev. Biochem. 45:747-803.

12. Goldberg, A. L, R. Voelimy, and K. H. SreedharaSwamy. 1979. In H. Holzer and H. Tschesche (ed.),Studies of the pathway for protein degradation in E.coli and mammalian cells, p. 35-48. Springer-Verlag,Berlin-Heidelberg.

13. Gorenstein, C., and J. R. Warner. 1977. Synthesis andturnover of ribosomal proteins in the absence of 60Ssubunit assembly in Saccharomyces cerevisiae. Mol.Gen. Genet. 157:327-332.

14. Hansen, M. T., M. L Pato, S. Molin, N. P. Fiil, and K.von Meyenburg. 1975. Simple downshift and resultinglack of correlation between ppGpp pool size and ribo-nucleic acid accumulation. J. Bacteriol. 122:585-591.

15. Irr, J. D. 1972. Control of nucleotide metabolism andribosomal ribonucleic acid synthesis during nitrogenstarvation of Escherichia coli. J. Bacteriol. 110:554-561.

16. Kerr, S. E., and K. Seraidarian. 1945. The separationof purine nucleosides from free purines and the deter-mination of the purines and ribose in these fractions. J.Biol. Chem. 159:211-225.

17. Mandelstam, J. 1960. The intracellular turnover of pro-tein and nucleic acids and its role in biochemical differ-entiation. Bacteriol. Rev. 24:289-308.

18. Maruyama, H., and D. Mizuno. 1970. Ribosome degra-dation and the degradation products in starved Esche-richia coli. I. Comparison of the degradation rate andof the nucleotide pool between Escherichia coli B andQ-13 strains in phosphate deficiency. Biochim. Biophys.Acta 199:159-165.

19. McCarthy, B. J. 1962. The effects of magnesium starva-tion on the ribosome content of Escherichia coli.Biochim. Biophys. Acta 55:880-888.

20. Natori, S., R. Nozawa, and D. Mizuno. 1966. Theturnover of ribosomal RNA of Escherichia coli in amagnesium-deficient stage. Biochim. Biophys. Acta114:245-253.

21. Olsson, M. O., and L. A. Isaksson. 1979. Analysis ofrpsD mutations in Escherichia coli. III. Effects of rpsDmutations on expression of some ribosomal proteingenes. Mol. Gen. Genet. 169:271-278.

22. Pao, C. C., and J. Gallant. 1978. A gene involved in themetabolic control of ppGpp synthesis. Mol. Gen. Genet.158:271-278.

23. Pao, C. C., and J. Gallant. 1979. A new nucleotideinvolved in the stringent response in Escherichia coli.J. Biol. Chem. 254:688-692.

24. Pine, M. J. 1972. Turnover of intracellular proteins. Annu.Rev. Microbiol. 26:103-126.

25. Rafaeli-Eshkol. D., D. Epstein, and A. Hershko. 1974.

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Roles of protein synthesis and tRNA aminoacylation inthe regulation of intracellular protein breakdown in E.coli. Biochem. Biophys. Res. Commun. 61:899-905.

26. Rafaeli-Eshkol, D., and A. Hershko. 1974. Regulationof intracellular protein breakdown in stringent and re-laxed strains of E. coli. Cell 2:31-35.

27. Stanley, P. E., and S. G. Williams. 1969. Use of theliquid scintillation spectrometer for determining aden-osine triphosphate by the luciferase enzyme. Anal. Bio-chem. 29:381-392.

28. St. John, A. C., K. Conklin, E. Rosenthal, and A. L.Goldberg. 1978. Further evidence for the involvementof charged tRNA and guanosine tetraphosphate in thecontrol of protein degradation in Escherichia coli. J.Biol. Chem. 253:3945-3951.

29. St. John, A. C., and A. L Goldberg. 1978. Effects ofreduced energy production on protein degradation, gua-nosine tetraphosphate, and RNA synthesis in Esche-richia coli. J. Biol. Chem. 253:2705-2711.

30. Sussman, A. J., and C. Gilvarg. 1969. Protein tumoverin amino acid-starved strains of Escherichia coli K-12

differing in their ribonucleic acid control. J. Biol. Chem.244:6304-6308.

31. Voellmy, R1, and A. L. Goldberg. 1980. Guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and the regula-tion of protein breakdown in Escherichia coli. J. Biol.Chem. 255:1008-1014.

32. Wallace, B. J., and B. D. Davis. 1973. Cyclic blockadeof initiation sites by streptomycin-damaged ribosomesin Escherichia coli: an explanation for dominance ofsensitivity. J. Mol. Biol. 75:377-390.

33. Warner, J. R. 1977. In the absence of ribosomal RNAsynthesis, the ribosomal proteins of HeLa cells aresynthesized normally and degraded rapidly. J. Mol.Biol. 115:315-333.

34. Willets, N. S. 1967. Intracellular protein breakdown innon-growing cells of Escherichia coli. Biochem. J. 103:453-461.

35. Zimmerman, R. A. 1974. RNA-protein interactions inthe ribosome, p. 225-269. In M. Nomura, A. Tissieres,and P. Lengyel (ed.), Ribosomes. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

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