- Email: [email protected]
Degradation of intracellular protein in Salmonella typhimurium peptidase mutants
J. Mol. Biol.
(1980) 143, 21-33
Degradation of Intracellular Protein in Salmonella typhimurium Peptidase Mutants CAREEN
LOUIS
YEN,
GREES
AND CHARLES G. MILLER
Department of Microbiology Case Western Reserve UnGuersity Cleveland, Ohio 44106, U.S.B. (Received
28 January
1980. and in revised
form
16 Jun,e 1980)
Multiply peptidase-deficient mutant strains ofSalmonella typhimurium fail to carry out normal protein degradation during starvation for a carbon source. In these mutants, the extent of protein breakdown during starvation is about fourfold less than in the wild type. The products of protein breakdown in the mutant are mainly small, trichloroacetic acid-soluble peptides, not free amino acids as in the wild type.
The carbon-starved amino
acid from
starvation shows
mutant strain produces only about one thirtieth protein
as the wild
type.
As a result,
protein
as much free
synthesis
during
is reduced in the mutant compared to the wild type and the mutant strain
a greatly
prolonged
lag phase after
a nutritional
shift-down.
1. Introduction The occurrence of protein turnover in bacteria has been recognized for some time (Mandelstam, 1960; Pine, 1972). Most of the protein in Escherichita coli appears to be relatively stable during exponential growth but starvation for a variety of required nutrients can lead to extensive protein breakdown (Pine, 1972; Mandelstam. 19586: Willets, 1967). Presumably. starvation mimics a growth shift-down or diauxic lag during which new enzymes can be synthesized only from amino acids produced by turnover (Mandelstam, 1963). Although there have been numerous physiological studies of starvation-induced protein turnover in bacteria (see Pine, 1972; Goldberg & St John, 1976, for recent reviews) relatively little is known about the degradation pathway. Several proteases and peptidases that might function in such a pathway have been purified and characterized (see Miller, 1975a for a review) but it has not been possible to assign these enzymes specific roles in any proteolytic process. One approach to assigning a particular enzyme to a specific physiological process involves isolating mutants lacking the enzyme and determining the effect of loss of the enzyme on the process. Mutants missing proteolytic enzymes have so far provided only negative information about their physiological roles : strains lacking both proteases I and II carry out a variety of proteolytic processes as well as their wild-type parents (Miller et al., 1976; Heiman & Miller, 1978; Miller & Zipser, 1977). We have reported the isolation (Miller & MacKinnon, 1974; McHugh & Miller, 1974: 21 0022- 2836/80/290021-13
$02.00/O
0 1980 Academic Press Inc. (London) Ltd.
PI
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Miller & Schwartz. 1978) and genetic characterization (Miller, 19756) of other types typhimurium and E. coli mutants lacking several peptidases. These of Snlmonrlla strains were isolated k)y screening for the inability to hydrolyze a chromogenic prptidase substrate (mutants lacking peptidase ,U) or by penicillin s&&on for failure to use small peptides as amino acid sources (mutants lacking peptidases I). A. B. Q. t’). The enzymes missing in these mutants arc all aminopept,idases or dipeptidases and are known to funcbion in the catabolism of exogenously supplied peptides (Miller & MacKinnon. 1974: McHugh & Miller, 1971: Kirsh rt rrl.. 1978). \t’e wished
to
find
out
if
these
enzymes
also
participate
in
the
degradation
of
intracellular proteins elicited by carbon-source starvation. In t)his pa,per we report studies of carbon-starvation-induced protein degradation in wild-thy and prptidase-deficient rnuta,nt strains of S. typhin~urium.
2. Materials All bacterial strains Tahlr 1, The isolation
previously
and Methods
used were derived from S. and genetic characterization
(Miller 8 Mackinnon,
typhimuriu7n
of these
1,Td and are described in strains have been described
197-C:Miller. 197.56).Sevrxral steps in the construction
of the
multiply peptidasr-deficient strain required mutagenesis. In order to avoid any possibility that additional unknown mutations acquired as a result of mutagenesis might effect the phenotype ofthese strains. we hare constructed an isogenic series of strains in which all of the peptidase mutations ha\ve been transferred hy transduction to a common unmutagenized hackground. The construction of these strains relied heavily on recently developed rt 01.. 1977) and will be described elsewhere. The newI> translocon technology (Kleckner constructed multiply peptidase-deficient strain (TX843) contains the same pips p~p,J and prpR alleles as the originally isolated strain TX215 (see Table 1). Because the pryI> mutation of TX215 leads to the production of a partially active peptidase D (Kirsh rt (I/.. 1978), strain TN843 was constructed using pepDd. a non-leaky mutation (Kirsh rt al.. 1978). Strains containing only 1 ofthe 4 peptidases missing in TN215 or TX843 (referred to as “ 1 + ” st,rains) were constructed from the multiply peptidase-deficient parent strain hy transduction (selecting for peptide utilization) and t.he presence of t,hc expected peptidase was
Strain designation TN251 TN294 TN293 TN296 TN297 TN843
TN873 TN874 TX875 TN894
Lrn + transductant from TN21 3 (Miller h MacKinnon. 1974) From TS2.51 by transduction From TX251 by transduction From TN251 by transduction
pepA pepB1 pepD1 pF,rpS10 pcpBl prpLI1 prp,VlO prpA I prpD1 From TN251 hy transduction prp*VlO prpA I prpH1 transferred to unmutagenized p~pn;lO Papa I pepB1 prpD.3 prp mutations LT2 background (Green 8: Miller, unpublished data) l+om TX843 bg transduction p’pA 1 prpB1 prpLx3 From TN843 by transduction prpXl0 p~pK1 prpD.3 Prom TN843 by transduction prpx10 pepA 1 pPpD3 From TN843 by transduction prpSl0 pepA 1 pepB1
PK.O’I‘EIS
DEGKADATIOS
IS
SA LMOSELL.4
23
iWl’T.-\XTS
confirmed by a gel electrophoresis peptidase activity assay as described previously (Miller & MacKinnon, 1974). Media and growth conditions have been described previously (Miller 8r MacKinnon. 1974). Medium E lacking citrate (NCE medium) and containing 0.05 M-KC] was used as the carbonfree medium unless otherwise noted. (1,) I:r,rrtic Transduction
with
kh,n
ig7trs
phage P22 int-3 was performrd (c) Drtwmination
of growth
as described
of pro&in
(1970).
cold lag times
ratrs
(:rowth rates were determined br measuring the absorbance at spectrophotometrr. For determinaiion of lag times after shift-down minimal glucose medium, cells were grown in nutrient broth to centrifugation. washed twice in ice-cold saline and diluted 1 : 10 into containing 0,4’& glucose. (Comparable results were obtained when turr was used to wash t’he cells.) (d) Lktrrminatiorc
by Roth
600 nm using a (:ilford from nutrient broth t’o A,,,, = 0.2, collected 1)~. pre-warmed medium F: saline at room tempera~
dpgradatiott
The usual design of the experiments to determine the extent of breakdown of pre-existing cellular proteins by the release of trichloroacetic acid-soluble radioactivity is similar to procedures used by several other workers (Mandelstam. 195%; Schlessinger & Ben Hamida. 1966: Goldberg, 1972). Cells were labelled during growth by diluting an overnight culture (grown in minimal glucose medium) 1 : 100 into‘2.i ml of the same medium cont,aininp 0.1 &‘(I r,-[4,5-3H]leucine/ml (25 mC”i/nmol). After cultures had grown to d 600 = 0.2 (-2.5 h). unlabelled I,-leucine was added to a final concn of 300 pg/ml and incubation contjinued for an additional 60 min. Cells were pelleted by ventrifugation, washed twice in growth medium without glucose, and resuspended in 25 ml of carbon-free NCE medium. Samples (0.9 ml) were withdraw-n at various times into 50?,, trichloroacrtic acid (0.1 ml) and allowed to stand for at least I h at 4°C. The acid-insoluble precipitate was pelleted by centrifugation and O.l-ml samples of the supernatant fluid were taken for determination of radioactivity by liquid scintillation counting in Triton/t,oluenr scintillation fluid (Patterson & (ireen, 1965). The total incorporated radioactivity was determined by counting samples of the culture/trichloroacetic-acid mixture before centrifugation. Cell viability during carborr-starvation was monitored by plate counts on nutrient agar. (e) Phromatography
of acid-soluble
matwial
(‘ells wer(A grown, labelled. washed and starved as described above except that the specificact,irity of the [ 14(‘lleucinr was 1 O-fold greater. Samples (4.5 ml) of the starving culture were withdrawn into O..i ml 50 sr/btrichloroacetic acid and rrfrigcrated at 4°C for at least 1 h brforr, pelleting acid-insoluble material by centrifugation (20 min, 12.000 g). The acid-solublt~ fractions were lyophilized and resuspended in 0.5 to 2.25 ml of chromatography buffer A (280 ml acetic acid. 3 ml pyridine, 717 ml water). A sample (0.5 ml) of this material was c.hromatographed at 50°C on a 0.9 cm x 23 cm column of Durrum DC-I A cation-exchange resin, The column teas eluted with a quadratic gradient similar to that described by Vogt cutul. (1975). Mixing chambers 1 and 2 each contained 150 ml of buffer A: chamber 3 contained I50 ml of buffer R (189 ml acetic: acid, 108 ml pyridinr. 703 ml water). After each run, thv c.olumn was washed with 2 iv-pyridine before being rr-equilibrated with buffer A. Fractions (3.5 ml) were collected in scint,illation vials. dried in a forced air oven (100 to 120°C’). redissolved in 0.4 ml 0.01 M-H(‘1, and counted in Trit,on/toluencl scintillation fluid. The results arc plotted as a percentage of the total counts rluted WTSUS fraction number. Recovery of’ radioactivity applied to the column was -950/0 for the wild-type a.nd -65% for the cluadruplr mut,ant (TN843). Chromatography of a mixture of unlabelled acid-soluble material
24
(‘.
YEN.
L.
GKEES
AND
(‘.
(:.
MILLER
from the mutant and a known quantity of [‘4C]leucine showed that more than 95% of the leucine label could be recovered from the column. Thus the loss of label observed during chromatography of material from the mutant strain does not. affect the determination of the amount of free leucine produced during starvation. (f) Measurement
of protein
synthesis
durkg
carbon -starvation
Cultures were grown in minimal glucose medium to A6,,,, = 0.3, washed twice in carbonfree NCE medium, and resuspended in carbon-free medium containing [‘%]leucine (0.1 &i/ml, 10 $2i/nmol). Samples were removed and treated with trichloroacetic acid as described above; the precipitates were collected on Millipore filters (0.45 pm), washed with cold 5% acid, and dried for 1 h at 60°C. Radioactivity on the filters was determined by liquid scintillation counting.
3. Results (a) Protein, degradation
in peptidase
mutants
The effect of peptidase deficiency on protein degradation during carbonstarvation is shown in Figure 1. In this experiment protein was labelled during exponential growth with radioactive leucine. Cells were harvested by centrifugation, resuspended in medium containing no utilizable carbon source, and the production of trichloroacetic acid-soluble protein breakdown products was followed. Neither the wild-type nor the mutant strain loses viability during the starvation period. The strain deficient in peptidases N, A, B and D clearly shows a reduced rate of protein degradation compared to the wild-type pep+ strain. The amount of acid-soluble material produced by the mutant during eight hours of starvation is approximately fourfold lower than in the wild-type strain. Protein degradation in strains containing on1 y one of the four peptidases missing in TN843 is shown in Figure 2 (these strains were constructed by transduction from TN843). Each of these strains shows increased production, relative to TN843, of acid-soluble radioactive material during starvation. These results show that all four of the peptidase activities missing in TN843 can participate in the process of protein
Time
(h)
FIG. 1. Protein degradation during starvation for a carbon source. -e-e--. -m--W-, TN843 (pepN- pepA- pepB- pepD-).
LT2 (wild-type):
PR ,OTEIN
DEGRADATION
IN
Time
FIG. pepBpepBpepB-
SALMONELLA
MUTA
NTS
25
(h)
2. Protein degradation during starvation for a carbon source. -@-a--, pepD-);-A---, TN894 (pepiT pepA- pepB- pepD+);-m-m---, pepD-): -O-O--, TN575 (pepN- papA pepB+ pepD-); --O-O-, pepD-).
TN873 (pepN+ pepA -TN874 (pcpN- pcy4 + TN843 (pepN- prprl~
breakdown induced by carbon-starvation. They do not, however, provide an accurate estimate of the quantitative significance of these enzymes in the breakdown process because, as discussed in the next section, measurement of the production of acid-soluble protein breakdown products in peptidase-deficient strains does not accurately measure the formation of free amino acids. (b) Products
of protein,
degradation,
in wild-type
and mutant strains
Previous studies of protein degradation in starving wild-type E. c&i strains have shown that the trichloroacetic acid-soluble products of degradation consist almost entirely of free amino acids (Mandelstam, 1958a; Pine, 1970). In strains deficient in peptidases, however, it seemed possible that protein degradation might result in the formation of acid-soluble peptides in place of or in addition to free amino acids. To investigate this possibility we have characterized the acid-soluble degradation products in both wild-type and peptidase-deficient strains. In order to compare the acid-soluble degradation products in wild-type and mutant strains, cells labelled with radioactive leucine (i4C for wild-type, 3H for the strain missing four peptidases) during growth were starved for a carbon source for eight hours, samples of the acid-soluble material extracted from the two cultures were combined and subjected to gel filtration on Sephadex G25. The elution profile from this column (Fig. 3) indicates that (1) the acid-soluble material present in the culture of the peptidase mutant is not the same as that found in the wild-type, and (2) much of the material in the mutant strain seems to be larger than that found in the wild-type. If material other than free leucine is being produced in the mutant strain during carbon-starvation, the effect of peptidase loss on the physiologically important process of amino acid production has been underestimated in comparisons based on determinations of acid-soluble material (such as that made in Figs 1 and 2). To compare t,he abilities of mutants and wild-type strains to produce free amino acids,
2ti
(‘.
YEN.
1,. GttEE1J
AS11
(‘.
C:. ~III~LEII
90
70 Fractmn
number
FIG. 3. Gel filtration chromatography of trichloroacetic-soluble products of protein degradation during carbon-starvation. Cultures (50 ml) were labelled during growth with [ 14C]- (mutant) or [‘H Ileucine (wild-type), starved for carbon for 8 h, and material soluble in 5’$ trichloroacetic acid collected. The acid was removed by evaporation and the residue suspended in I M-acetic acid. The samples were rombined and chromatographrd on a I ,5 cm x 90 cm column of Sephadrx (:25 (Pharmacia Fine C”hvmicals, Inc.). equilibrated and eluted with 1 M-acetic acid. I-ml fractions were collected. -e-a-. jH (LT2): -O-O-. ‘% (TN251): arrows indicate the elution position of blue drxtran (ED). (:Iv-I,r,u-I,ru-(:l) (a),
and
leucinr
(L).
the following experiment was performed. Trichloroacetic acid-soluble radioactive material produced during starvation from both mutant and wild-type strains was chromatographed on a cation-exchange resin. Chromatography of material from the wild-type strain gave only one radioactive peak (Fig. 4(a)). This material eluted from the column in the same fractions as L-leucine (present as carrier and identified by reaction with ninhydrin). Chromatography of acid-soluble material from the mutant strain (Fig. 4(b)) revealed a heterogeneous mixture of labelled material. Because the free leucine peak is well-resolved in this mixture we could determine how much of the acid-soluble radioactive material produced in the mutant strain is free leucine by chromatographing the acid-soluble products collected at various times. Since we also know how much of the labelled protein has been converted to acid-soluble material at any time, we can calculate the fraction of labelled protein that has been degraded completely to yield free leucine. the physiologically significant protein degradation end product. Figure 5 presents the results of these calculations. The rate of production of free leucine from labelled protein (calculated from the slopes of the lines in Fig. 5) is approximately 30-fold lower in the multiple mutant than in the wild-type strain. Using the same procedures, we can estimate the quantitative significance of each of the peptidases absent in the multiple mutant.
Fraction
number
FIG:. 4. Ion-exchange chromatography of trichloroacetic acid-soluble Acid-soluble breakdownproductsfrom (a) LTP (wild-type) and (b) TN843 were collected after 2 h of carbon-source starvation and chromatographed ,XIethods. ln (a) no fractions other than 59. 60 and 61 contained >O~l”,
protein breakdown prodwts (pepS- prpil- prpR- prpf~ ) LS described in Materials and of the total radioactivity.
The fraction of protein converted to free leucine after two hours of carbonstarvation is presented in Table 2. These results show that the capacity of the multiply peptidase-deficient strain to produce free amino acids from protein during carbon-starvation is drastically reduced in comparison with the wild-type. They aIso show that measurements ofthe formation of trichloroacetic acid-soluble protein breakdown products do not provide an accurate assessment of the production of free amino acids from protein
28
C. YEN,
L.
GREEN
AND
(‘.
(:.
MILLER
Time (h)
FIG. 5. Production of free leueine during carbon-source starvation. -e-m--. LT2 (wild-type): acid-soluble degradation -m-m-, TN843 (pepA- pepA- pepR- pepL)-). The trichloroacetic products were collected at various times during carbon-starvation, subjected to ion-exchange chromatography (see Fig. 4), and the fraction of free leucine determined. Free Ieucine was the only labelled compound present in the trichloroacetic acid extracts ofthe wild type at all the times tested. The fraction of the total radioactivity in protein that has been converted to free leucine at any time was calculated from the relationship: (fraction free leucine),=(fraction of the acid-soluble cts/min in free leucine), x (fraction of the total incorporated cts/min that is acid-soluble),.
TAHLE 2 Free amino acid production
carbon-starvation
Peptidase genotype P’PPA PePR
Strain LT2 TN843 TN873 TN874 TN875 TN894
during
+ + -
+ + -
+ + -
+ +
7.1 0.3 5.4 5.1 I s-3 2.7
because the acid-soluble products of protein breakdown in the multiply mutant strain appear to be a heterogeneous mixture of small peptides rather than free amino acids. These results also provide information on the relative efficiency with which each one of the four peptidases missing in the multiple mutant can function in the absence of the other three. The data in Table 2 show that none of the four peptidases can alone restore free leucine production to wild-type levels. The specificities of these peptidases have been studied using in vitro assays as well as by characterizing the peptide utilization patterns of strains with three peptidase mutations. The results of these studies (Miller & MacKinnon, 1974; Kirsh et al., 1978) indicate that these peptidases have broadly overlapping specificities toward the substrates
PROTEIN
DEGRADATION
IS
SALMOA’ELLA
MMlTTASTS
29
studied. Peptidases N and A hydrolyze a variety of dipeptides, dipeptide amides. tripeptides and tetrapeptides. Peptidase D cleaves only dipeptides, most of which are also hydrolyzed by peptidase N and A. Peptidase B hydrolyzes both di- and tripeptides but its specificity does not seem to be as broad as that of the other three enzymes. The data of Table 2 suggest that at least some of the overlapping specificities displayed toward exogenously supplied peptides is also shown toward peptides generated inside the cell. The strains containing peptidase 11’ (TN873) or peptidase A (TN874) each produce greater than 50y0 of the wild-type levels of free leucine. These two enzymes must, therefore, have at least partially overlapping specificities t’oward the peptides generated during carbon-starvation. In addition. t’he results with the pepD+ strain suggest that a significant fraction ( - 359,;) of the peptides that accumulate in the multiple mutant are dipeptides. (c) Protein
synthesis
during
starvation
During starvation no net protein synthesis is possible but amino acid incorporation can occur as a result of protein turnover (Mandelstam, 1958a,b). Amino acids for the synthesis of new proteins are provided by protein degradation. Since the production of amino acids during starvation is sharply reduced in the peptidasedeficient strains, we expected that) the rate of protein synthesis during starvation would also be reduced in the peptidase mutants. To test this expectation, we measured the rate of protein synthesis during starvation for carbon in peptidasedeficient and wild-type strains (Fig. 6). Clearly the simultaneous absence of peptidases N, A. Band D (TN843) leads to a significant decrease in the ability of the cell to s.vnthesize protein during starvation. (d) Growth
charactrristks
of peptidase
mutants
Willets (1967) showed that in E. coli the transfer ofcells growing in a rich medium to minimal medium leads to an immediate increase in protein degradation. This increased degradation presumably provides amino acids for the synthesis of new prot,eins required for the resumption of growth. Since the peptidase-deficient strains show diminished abilities to degrade and synthesize protein during carbon starvation, they might be expected to show similar deficiencies in a shift-down and. as a result, they might have prolonged lag times after such a shift. Figure 7 shows growth curves for the wild-type and a multiply peptidase-deficient mutant. When the cells were shifted from nutrient broth to minimal glucose medium the wild-type showed a lag time of about 60 minutes before resuming exponential growth. The pepNABD strain, TN843. requires more than eight hours for resumpt,ion of growth. (Downshifted cultures of TN843 grow up after overnight, (- 15 h) incubation.) When the broth-grown cells were resuspended in minimal medium supplemented with the 20 r,-amino acids (at 1 x 10m4 M), no lag was observed in LT2. The mutant strain’s lag period was reduced to -30 minutes, Strains containing only one of the four peptidases missing in the pepN pepA pepB pepD strain show lag times intermediate between the mutant and the wild type (Table 3). These results strongly indicate that the observed increase in lag time is caused by the diminished availability of amino acids which results from the combined loss of peptidases N\‘. A, B and D.
(‘.
30
YES,
I,
GREES
I
2
AND
(‘.
(:.
MILLER
4
3
5
Time (h)
kc:. TX873 TX894 TN843
6. Protein synthesis during rarbon~sourw (pepAr+ prp-4 - ppH- pepD-): -U-E+> (prpS- pep.4 - PP~H- p~pD+): -O-o--. (p+Vprpd - pel-‘R- prpI)-).
Doubling
times
starvation. -b-n-, LT:! (wild-type): TN871 (f~irp,.L’~pcpA+ prpB- pepD-); TN875 @p-V- fqut p~pR+ pq~D-);
nn.d lag times of wild-typ
and
mutant
-A-A-, -m-m--. [email protected].
straitts Doubling timr: (min)
LT2 TN843 TN873 TN874 TN8i5 TN894
+ + -
+ + -
+ + -
+ +
70 >4HO 100 70 90 100
56 x0 TO 62 70
70
t Measured after shift from nutrient brot,h to minimal ghwose medium. These times can be reproduced to about _+IS”,. The lag times of the strains with 3 peptidase mutations may not differ significantly from Lx?. 1 In minimal glucose medium.
Doubling times for a series of strains containing various peptidase mutations have also been determined (Table 3). Multiple peptidase deficiency leads to a decreased exponential growth rate as well as prolonged lag times. This observation suggests that these enzymes play some role during exponential growth as well as during starvation.
l’K.OTEIN
DE(:KAD.ATIOS
0.4 -
IN
S/l LXO~VELLrZ
hll’T.AN’l’S
3I
(a)
0.3
-
0.2
-
O-l 008
-
0.06
-
0.04
. I
0.02 -
.
l
lb
-
.
.
.
-
E c z ;; E : G 51
0.01
-
o-5 04
-
I
I
240
300
I
I
I
60
120
1
I
(b)
0.3 o-2
0.1 0.08 0.06 0.04 7
2
0’02
I
180
360
420
Time (min)
PK:. 7. Growth after nutritional shift-down. Ckils growing exponentially in nutrient broth wcw washed and resuspended in minimal glucose medium (a) or minimal glucose medium containing the 20 standard I.-amino acids, each at 0.1 mM (h). -@-a--. LT2 (wild-type); -A-A-, TN843 (prp,V pe~1.1~
pepK prpr). 4. Discussion Each of the peptidases missing in the mutant strains studied in this paper can function in the catabolism of exogenously supplied peptides (Miller & MacKinnon. 1974: Miller & Schwartz, 1978; Kirsh et al., 1978). The major question we set out to answer was : do these peptidases also act in the degradation of intracellular protein during starvation 2 The results presented here indicate that they do function in carbon-starvation-induced protein degradation because in their absence (1) the extent of such degradation is decreased, and (2) the products of degradation are different The production ofpeptides by the starved mutant rather than free amino acids
3’2
('. YEN.
L. GREEK
AND
('. (:. MILLER
as in the wild type, indicates that these enzymes lie on the starvation degradation pathway. The most obvious hypothesis is that these enzymes function late in the degradation pathway, hydrolyzing small (trichloroacetic acid-soluble) peptides produced by the attack of other enzymes on larger polypeptides. If this is true. it is not immediately obvious why the rate of production of acid-soluble material is reduced in the mutant. Vl’e have considered three explanations for this observation. (1) Some of the polypeptides normally attacked by the peptidases missing in the mutant are acid-insoluble. These polypeptides might be large fragments produced by other hydrolytic enzymes or they might be native proteins that are normally degraded uia initial N-terminal aminopeptidase attack. In preliminary experiments employing gel filtration (Sephadex G75 in sodium dodecyl sulfate-cont’aining buffers) of sodium dodecyl sulfate-solubilized starved cells we found no evidence for the presence of a significant amount of labelled material of a size expected for peptides slightly too large to be acid-soluble in either the mutant or the wild-type. If the reduction in the amount of acid-soluble material observed in the mutant resulted from accumulation of material in a pool of peptides slightly larger than those detected as acid-soluble. we would expect as much as 15 t)o 20% of the total protein label to appear in this pool. (2) A second possible explanation for the observed reduction is that the peptides themselves might directly inhibit the action of other peptidases acting earlier in the degradation pathway. We know that the most deficient mutant studied here still contains several peptidase activities (Miller $ MacKinnon, 1974: McHugh & Miller, 1974: Yaron et al., 1972). At least, one of the peptidases still present in our mutant strain is known to be inhibited by di- and tripeptides (Yaron. 1974). (3) It is also possible that the reduction in the extent of formation of acid-soluble protein fragment,s during carbon-starvation is an indirect result of peptidase loss. Several lines of evidence indicate that protein degradation requires metabolic energy. St clohn & (Goldberg (1978) have proposed that in E. coli the intracellular ATT’ concentration must remain above a minimal level for protein turnover to occur. If metabolism of the amino acids produced by turnover makes a significant contribution to the energy supply of the cell during carbon-starvation, the failure of the mutants to produce free amino acids at the wild-type rate might result in a decrease in cellular ATP concentration. As a result, the initial steps of protein breakdown might not take place at the normal rate. The data available do not allow us to distinguish among these possibilities. The results of the turnover studies on strains containing only one of the four missing peptidases indicate that these peptidases show some overlap in their specificities toward peptides generated internally just as they do toward those supplied exogenously. Studies of peptide utilization by the strains having only one of the four peptidases have indicated that most small peptides can be hydrolyzed efficiently by more than one of these enzymes (Kirsh et al., 1978; Green, unpublished results). The large increase in the production of free leucine in these strains (particularly pepN+ or pepA+) compared to the quadruple mutant suggests that many of the peptides generated during turnover can be hydrolyzed by more than one of the missing peptidases. In the absence of a group of peptidases, protein turnover during starvation is blocked at a late step in which small peptides are usually converted to free amino
PROTEIS
DEGRADATION
IN
SA 1,:1fO.\‘\‘ELLd
Ml’TAX’I’S
33
acids. As a result of this block, starving mutant cells cannot synthesize protein at’ wild-type rates and growth lag times after a nutritional shift are greatly prolonged. These results support Mandelstam’s (1963) proposal that protein turnover during starvation constitutes an important mechanism of adaptation in bacteria. They also establish the importance of peptide-hydrolyzing enzymes in the protein turnover process. This work was supported by grants from the United States Public Health Service. by Research (lareer Development Award GM07321 from the National Institute for General Medical Sciences (to C. M.), and by Research grant AT1 0333 from the National Institute of Allergy and Infectious Disease. REFERENCES Goldberg, A. L. (1972). Proc. Nat. Acad. &i..lT.S.A. 69, 422-426. Goldberg. A. L. 8: St John, A. C. (1976). Annu. Rev. Biochem. 45, 747-803. Heiman. C. & Miller, C. G. (1978). J. Bacterial. 135, 588-594. Kirsh. M.. Dembinski, D. R., Hartman, P. E. &Miller, C. G. (1978). J. Bacterial. 134,36lL374. Kleckner. N., Roth, J. & Botstein, D. (1977). J. ~lilol. Biol. 116, 125159. Mandelstam, J. (1958a). Biochem. J. 69, 1033110. Mandelstam, J. (1958b). B&hem. J. 69, 116119. Mandelstam, J. (1960). Bactertil. Rev. 24, 289-308. Mandelstam, J. (1963). Ann. N.Y. Acud. Sci. 102, 621-636. McHugh, G. L. & Miller, C. G. (1974). J. Bacterial. 120, 364-371. Miller, C. G. (1975a). Annu. Rev. Microbial. 29, 485.504. Miller. C. G. (1975b). J. Bacterial. 122, 171-176. Miller. C. (:. & MacKinnon, K. (1974). J. Bacterial. 120, 355363. Miller, C. G. & Schwartz, G. (1978). J. Bucteriol. 135, 6033611. Miller, C. (:. 62 Zipser, D. (1977). J. Bactrriol. 130, 347-353. Miller, (1. G., Heiman, C. & Yen, C. (1976). J. Bacterial. 127, 490-497. Patterson, &I. S. & Greene, R. C. (1965). Anal. Chem. 37, 854-857. Pine, M. J. (1972). Annu. Rev. Microbial. 26, 103-126. Roth, ,J. R. (1970). In Methods of Enzymology (Tabor. H. & Tabor. C. W.. eds). vol. 17a, pp. 2235, Academic Press Inc., New York. Schlrssinger, D. & Ben-Hamida, F. (3966). Biochim. Biophys. Actu, 119, 171L182. St John, A. C. 8: Goldberg, A. L. (1978). J. Biol. Chem. 253, 27052711. Yogt, V. M., Eisenman, R. & Diggelmann, H. (1975). J. Mol. Riol. 96, 471-493. Willets, N. S. (1967). Biochem. J. 103, 4533461. Yaron. A. (1974). Israel J. Chem. 12, 651-662. Yaron. A., Mlynar, D. & Berger, A. (1972). Biochem. Biophy. Res. (‘ommun. 47, 897-902.
(1980) 143, 21-33
Degradation of Intracellular Protein in Salmonella typhimurium Peptidase Mutants CAREEN
LOUIS
YEN,
GREES
AND CHARLES G. MILLER
Department of Microbiology Case Western Reserve UnGuersity Cleveland, Ohio 44106, U.S.B. (Received
28 January
1980. and in revised
form
16 Jun,e 1980)
Multiply peptidase-deficient mutant strains ofSalmonella typhimurium fail to carry out normal protein degradation during starvation for a carbon source. In these mutants, the extent of protein breakdown during starvation is about fourfold less than in the wild type. The products of protein breakdown in the mutant are mainly small, trichloroacetic acid-soluble peptides, not free amino acids as in the wild type.
The carbon-starved amino
acid from
starvation shows
mutant strain produces only about one thirtieth protein
as the wild
type.
As a result,
protein
as much free
synthesis
during
is reduced in the mutant compared to the wild type and the mutant strain
a greatly
prolonged
lag phase after
a nutritional
shift-down.
1. Introduction The occurrence of protein turnover in bacteria has been recognized for some time (Mandelstam, 1960; Pine, 1972). Most of the protein in Escherichita coli appears to be relatively stable during exponential growth but starvation for a variety of required nutrients can lead to extensive protein breakdown (Pine, 1972; Mandelstam. 19586: Willets, 1967). Presumably. starvation mimics a growth shift-down or diauxic lag during which new enzymes can be synthesized only from amino acids produced by turnover (Mandelstam, 1963). Although there have been numerous physiological studies of starvation-induced protein turnover in bacteria (see Pine, 1972; Goldberg & St John, 1976, for recent reviews) relatively little is known about the degradation pathway. Several proteases and peptidases that might function in such a pathway have been purified and characterized (see Miller, 1975a for a review) but it has not been possible to assign these enzymes specific roles in any proteolytic process. One approach to assigning a particular enzyme to a specific physiological process involves isolating mutants lacking the enzyme and determining the effect of loss of the enzyme on the process. Mutants missing proteolytic enzymes have so far provided only negative information about their physiological roles : strains lacking both proteases I and II carry out a variety of proteolytic processes as well as their wild-type parents (Miller et al., 1976; Heiman & Miller, 1978; Miller & Zipser, 1977). We have reported the isolation (Miller & MacKinnon, 1974; McHugh & Miller, 1974: 21 0022- 2836/80/290021-13
$02.00/O
0 1980 Academic Press Inc. (London) Ltd.
PI
(‘.
YEN.
12. (:IIEES
ASl)
(’
c:.
MILLEK
Miller & Schwartz. 1978) and genetic characterization (Miller, 19756) of other types typhimurium and E. coli mutants lacking several peptidases. These of Snlmonrlla strains were isolated k)y screening for the inability to hydrolyze a chromogenic prptidase substrate (mutants lacking peptidase ,U) or by penicillin s&&on for failure to use small peptides as amino acid sources (mutants lacking peptidases I). A. B. Q. t’). The enzymes missing in these mutants arc all aminopept,idases or dipeptidases and are known to funcbion in the catabolism of exogenously supplied peptides (Miller & MacKinnon. 1974: McHugh & Miller, 1971: Kirsh rt rrl.. 1978). \t’e wished
to
find
out
if
these
enzymes
also
participate
in
the
degradation
of
intracellular proteins elicited by carbon-source starvation. In t)his pa,per we report studies of carbon-starvation-induced protein degradation in wild-thy and prptidase-deficient rnuta,nt strains of S. typhin~urium.
2. Materials All bacterial strains Tahlr 1, The isolation
previously
and Methods
used were derived from S. and genetic characterization
(Miller 8 Mackinnon,
typhimuriu7n
of these
1,Td and are described in strains have been described
197-C:Miller. 197.56).Sevrxral steps in the construction
of the
multiply peptidasr-deficient strain required mutagenesis. In order to avoid any possibility that additional unknown mutations acquired as a result of mutagenesis might effect the phenotype ofthese strains. we hare constructed an isogenic series of strains in which all of the peptidase mutations ha\ve been transferred hy transduction to a common unmutagenized hackground. The construction of these strains relied heavily on recently developed rt 01.. 1977) and will be described elsewhere. The newI> translocon technology (Kleckner constructed multiply peptidase-deficient strain (TX843) contains the same pips p~p,J and prpR alleles as the originally isolated strain TX215 (see Table 1). Because the pryI> mutation of TX215 leads to the production of a partially active peptidase D (Kirsh rt (I/.. 1978), strain TN843 was constructed using pepDd. a non-leaky mutation (Kirsh rt al.. 1978). Strains containing only 1 ofthe 4 peptidases missing in TN215 or TX843 (referred to as “ 1 + ” st,rains) were constructed from the multiply peptidase-deficient parent strain hy transduction (selecting for peptide utilization) and t.he presence of t,hc expected peptidase was
Strain designation TN251 TN294 TN293 TN296 TN297 TN843
TN873 TN874 TX875 TN894
Lrn + transductant from TN21 3 (Miller h MacKinnon. 1974) From TS2.51 by transduction From TX251 by transduction From TN251 by transduction
pepA pepB1 pepD1 pF,rpS10 pcpBl prpLI1 prp,VlO prpA I prpD1 From TN251 hy transduction prp*VlO prpA I prpH1 transferred to unmutagenized p~pn;lO Papa I pepB1 prpD.3 prp mutations LT2 background (Green 8: Miller, unpublished data) l+om TX843 bg transduction p’pA 1 prpB1 prpLx3 From TN843 by transduction prpXl0 p~pK1 prpD.3 Prom TN843 by transduction prpx10 pepA 1 pPpD3 From TN843 by transduction prpSl0 pepA 1 pepB1
PK.O’I‘EIS
DEGKADATIOS
IS
SA LMOSELL.4
23
iWl’T.-\XTS
confirmed by a gel electrophoresis peptidase activity assay as described previously (Miller & MacKinnon, 1974). Media and growth conditions have been described previously (Miller 8r MacKinnon. 1974). Medium E lacking citrate (NCE medium) and containing 0.05 M-KC] was used as the carbonfree medium unless otherwise noted. (1,) I:r,rrtic Transduction
with
kh,n
ig7trs
phage P22 int-3 was performrd (c) Drtwmination
of growth
as described
of pro&in
(1970).
cold lag times
ratrs
(:rowth rates were determined br measuring the absorbance at spectrophotometrr. For determinaiion of lag times after shift-down minimal glucose medium, cells were grown in nutrient broth to centrifugation. washed twice in ice-cold saline and diluted 1 : 10 into containing 0,4’& glucose. (Comparable results were obtained when turr was used to wash t’he cells.) (d) Lktrrminatiorc
by Roth
600 nm using a (:ilford from nutrient broth t’o A,,,, = 0.2, collected 1)~. pre-warmed medium F: saline at room tempera~
dpgradatiott
The usual design of the experiments to determine the extent of breakdown of pre-existing cellular proteins by the release of trichloroacetic acid-soluble radioactivity is similar to procedures used by several other workers (Mandelstam. 195%; Schlessinger & Ben Hamida. 1966: Goldberg, 1972). Cells were labelled during growth by diluting an overnight culture (grown in minimal glucose medium) 1 : 100 into‘2.i ml of the same medium cont,aininp 0.1 &‘(I r,-[4,5-3H]leucine/ml (25 mC”i/nmol). After cultures had grown to d 600 = 0.2 (-2.5 h). unlabelled I,-leucine was added to a final concn of 300 pg/ml and incubation contjinued for an additional 60 min. Cells were pelleted by ventrifugation, washed twice in growth medium without glucose, and resuspended in 25 ml of carbon-free NCE medium. Samples (0.9 ml) were withdraw-n at various times into 50?,, trichloroacrtic acid (0.1 ml) and allowed to stand for at least I h at 4°C. The acid-insoluble precipitate was pelleted by centrifugation and O.l-ml samples of the supernatant fluid were taken for determination of radioactivity by liquid scintillation counting in Triton/t,oluenr scintillation fluid (Patterson & (ireen, 1965). The total incorporated radioactivity was determined by counting samples of the culture/trichloroacetic-acid mixture before centrifugation. Cell viability during carborr-starvation was monitored by plate counts on nutrient agar. (e) Phromatography
of acid-soluble
matwial
(‘ells wer(A grown, labelled. washed and starved as described above except that the specificact,irity of the [ 14(‘lleucinr was 1 O-fold greater. Samples (4.5 ml) of the starving culture were withdrawn into O..i ml 50 sr/btrichloroacetic acid and rrfrigcrated at 4°C for at least 1 h brforr, pelleting acid-insoluble material by centrifugation (20 min, 12.000 g). The acid-solublt~ fractions were lyophilized and resuspended in 0.5 to 2.25 ml of chromatography buffer A (280 ml acetic acid. 3 ml pyridine, 717 ml water). A sample (0.5 ml) of this material was c.hromatographed at 50°C on a 0.9 cm x 23 cm column of Durrum DC-I A cation-exchange resin, The column teas eluted with a quadratic gradient similar to that described by Vogt cutul. (1975). Mixing chambers 1 and 2 each contained 150 ml of buffer A: chamber 3 contained I50 ml of buffer R (189 ml acetic: acid, 108 ml pyridinr. 703 ml water). After each run, thv c.olumn was washed with 2 iv-pyridine before being rr-equilibrated with buffer A. Fractions (3.5 ml) were collected in scint,illation vials. dried in a forced air oven (100 to 120°C’). redissolved in 0.4 ml 0.01 M-H(‘1, and counted in Trit,on/toluencl scintillation fluid. The results arc plotted as a percentage of the total counts rluted WTSUS fraction number. Recovery of’ radioactivity applied to the column was -950/0 for the wild-type a.nd -65% for the cluadruplr mut,ant (TN843). Chromatography of a mixture of unlabelled acid-soluble material
24
(‘.
YEN.
L.
GKEES
AND
(‘.
(:.
MILLER
from the mutant and a known quantity of [‘4C]leucine showed that more than 95% of the leucine label could be recovered from the column. Thus the loss of label observed during chromatography of material from the mutant strain does not. affect the determination of the amount of free leucine produced during starvation. (f) Measurement
of protein
synthesis
durkg
carbon -starvation
Cultures were grown in minimal glucose medium to A6,,,, = 0.3, washed twice in carbonfree NCE medium, and resuspended in carbon-free medium containing [‘%]leucine (0.1 &i/ml, 10 $2i/nmol). Samples were removed and treated with trichloroacetic acid as described above; the precipitates were collected on Millipore filters (0.45 pm), washed with cold 5% acid, and dried for 1 h at 60°C. Radioactivity on the filters was determined by liquid scintillation counting.
3. Results (a) Protein, degradation
in peptidase
mutants
The effect of peptidase deficiency on protein degradation during carbonstarvation is shown in Figure 1. In this experiment protein was labelled during exponential growth with radioactive leucine. Cells were harvested by centrifugation, resuspended in medium containing no utilizable carbon source, and the production of trichloroacetic acid-soluble protein breakdown products was followed. Neither the wild-type nor the mutant strain loses viability during the starvation period. The strain deficient in peptidases N, A, B and D clearly shows a reduced rate of protein degradation compared to the wild-type pep+ strain. The amount of acid-soluble material produced by the mutant during eight hours of starvation is approximately fourfold lower than in the wild-type strain. Protein degradation in strains containing on1 y one of the four peptidases missing in TN843 is shown in Figure 2 (these strains were constructed by transduction from TN843). Each of these strains shows increased production, relative to TN843, of acid-soluble radioactive material during starvation. These results show that all four of the peptidase activities missing in TN843 can participate in the process of protein
Time
(h)
FIG. 1. Protein degradation during starvation for a carbon source. -e-e--. -m--W-, TN843 (pepN- pepA- pepB- pepD-).
LT2 (wild-type):
PR ,OTEIN
DEGRADATION
IN
Time
FIG. pepBpepBpepB-
SALMONELLA
MUTA
NTS
25
(h)
2. Protein degradation during starvation for a carbon source. -@-a--, pepD-);-A---, TN894 (pepiT pepA- pepB- pepD+);-m-m---, pepD-): -O-O--, TN575 (pepN- papA pepB+ pepD-); --O-O-, pepD-).
TN873 (pepN+ pepA -TN874 (pcpN- pcy4 + TN843 (pepN- prprl~
breakdown induced by carbon-starvation. They do not, however, provide an accurate estimate of the quantitative significance of these enzymes in the breakdown process because, as discussed in the next section, measurement of the production of acid-soluble protein breakdown products in peptidase-deficient strains does not accurately measure the formation of free amino acids. (b) Products
of protein,
degradation,
in wild-type
and mutant strains
Previous studies of protein degradation in starving wild-type E. c&i strains have shown that the trichloroacetic acid-soluble products of degradation consist almost entirely of free amino acids (Mandelstam, 1958a; Pine, 1970). In strains deficient in peptidases, however, it seemed possible that protein degradation might result in the formation of acid-soluble peptides in place of or in addition to free amino acids. To investigate this possibility we have characterized the acid-soluble degradation products in both wild-type and peptidase-deficient strains. In order to compare the acid-soluble degradation products in wild-type and mutant strains, cells labelled with radioactive leucine (i4C for wild-type, 3H for the strain missing four peptidases) during growth were starved for a carbon source for eight hours, samples of the acid-soluble material extracted from the two cultures were combined and subjected to gel filtration on Sephadex G25. The elution profile from this column (Fig. 3) indicates that (1) the acid-soluble material present in the culture of the peptidase mutant is not the same as that found in the wild-type, and (2) much of the material in the mutant strain seems to be larger than that found in the wild-type. If material other than free leucine is being produced in the mutant strain during carbon-starvation, the effect of peptidase loss on the physiologically important process of amino acid production has been underestimated in comparisons based on determinations of acid-soluble material (such as that made in Figs 1 and 2). To compare t,he abilities of mutants and wild-type strains to produce free amino acids,
2ti
(‘.
YEN.
1,. GttEE1J
AS11
(‘.
C:. ~III~LEII
90
70 Fractmn
number
FIG. 3. Gel filtration chromatography of trichloroacetic-soluble products of protein degradation during carbon-starvation. Cultures (50 ml) were labelled during growth with [ 14C]- (mutant) or [‘H Ileucine (wild-type), starved for carbon for 8 h, and material soluble in 5’$ trichloroacetic acid collected. The acid was removed by evaporation and the residue suspended in I M-acetic acid. The samples were rombined and chromatographrd on a I ,5 cm x 90 cm column of Sephadrx (:25 (Pharmacia Fine C”hvmicals, Inc.). equilibrated and eluted with 1 M-acetic acid. I-ml fractions were collected. -e-a-. jH (LT2): -O-O-. ‘% (TN251): arrows indicate the elution position of blue drxtran (ED). (:Iv-I,r,u-I,ru-(:l) (a),
and
leucinr
(L).
the following experiment was performed. Trichloroacetic acid-soluble radioactive material produced during starvation from both mutant and wild-type strains was chromatographed on a cation-exchange resin. Chromatography of material from the wild-type strain gave only one radioactive peak (Fig. 4(a)). This material eluted from the column in the same fractions as L-leucine (present as carrier and identified by reaction with ninhydrin). Chromatography of acid-soluble material from the mutant strain (Fig. 4(b)) revealed a heterogeneous mixture of labelled material. Because the free leucine peak is well-resolved in this mixture we could determine how much of the acid-soluble radioactive material produced in the mutant strain is free leucine by chromatographing the acid-soluble products collected at various times. Since we also know how much of the labelled protein has been converted to acid-soluble material at any time, we can calculate the fraction of labelled protein that has been degraded completely to yield free leucine. the physiologically significant protein degradation end product. Figure 5 presents the results of these calculations. The rate of production of free leucine from labelled protein (calculated from the slopes of the lines in Fig. 5) is approximately 30-fold lower in the multiple mutant than in the wild-type strain. Using the same procedures, we can estimate the quantitative significance of each of the peptidases absent in the multiple mutant.
Fraction
number
FIG:. 4. Ion-exchange chromatography of trichloroacetic acid-soluble Acid-soluble breakdownproductsfrom (a) LTP (wild-type) and (b) TN843 were collected after 2 h of carbon-source starvation and chromatographed ,XIethods. ln (a) no fractions other than 59. 60 and 61 contained >O~l”,
protein breakdown prodwts (pepS- prpil- prpR- prpf~ ) LS described in Materials and of the total radioactivity.
The fraction of protein converted to free leucine after two hours of carbonstarvation is presented in Table 2. These results show that the capacity of the multiply peptidase-deficient strain to produce free amino acids from protein during carbon-starvation is drastically reduced in comparison with the wild-type. They aIso show that measurements ofthe formation of trichloroacetic acid-soluble protein breakdown products do not provide an accurate assessment of the production of free amino acids from protein
28
C. YEN,
L.
GREEN
AND
(‘.
(:.
MILLER
Time (h)
FIG. 5. Production of free leueine during carbon-source starvation. -e-m--. LT2 (wild-type): acid-soluble degradation -m-m-, TN843 (pepA- pepA- pepR- pepL)-). The trichloroacetic products were collected at various times during carbon-starvation, subjected to ion-exchange chromatography (see Fig. 4), and the fraction of free leucine determined. Free Ieucine was the only labelled compound present in the trichloroacetic acid extracts ofthe wild type at all the times tested. The fraction of the total radioactivity in protein that has been converted to free leucine at any time was calculated from the relationship: (fraction free leucine),=(fraction of the acid-soluble cts/min in free leucine), x (fraction of the total incorporated cts/min that is acid-soluble),.
TAHLE 2 Free amino acid production
carbon-starvation
Peptidase genotype P’PPA PePR
Strain LT2 TN843 TN873 TN874 TN875 TN894
during
+ + -
+ + -
+ + -
+ +
7.1 0.3 5.4 5.1 I s-3 2.7
because the acid-soluble products of protein breakdown in the multiply mutant strain appear to be a heterogeneous mixture of small peptides rather than free amino acids. These results also provide information on the relative efficiency with which each one of the four peptidases missing in the multiple mutant can function in the absence of the other three. The data in Table 2 show that none of the four peptidases can alone restore free leucine production to wild-type levels. The specificities of these peptidases have been studied using in vitro assays as well as by characterizing the peptide utilization patterns of strains with three peptidase mutations. The results of these studies (Miller & MacKinnon, 1974; Kirsh et al., 1978) indicate that these peptidases have broadly overlapping specificities toward the substrates
PROTEIN
DEGRADATION
IS
SALMOA’ELLA
MMlTTASTS
29
studied. Peptidases N and A hydrolyze a variety of dipeptides, dipeptide amides. tripeptides and tetrapeptides. Peptidase D cleaves only dipeptides, most of which are also hydrolyzed by peptidase N and A. Peptidase B hydrolyzes both di- and tripeptides but its specificity does not seem to be as broad as that of the other three enzymes. The data of Table 2 suggest that at least some of the overlapping specificities displayed toward exogenously supplied peptides is also shown toward peptides generated inside the cell. The strains containing peptidase 11’ (TN873) or peptidase A (TN874) each produce greater than 50y0 of the wild-type levels of free leucine. These two enzymes must, therefore, have at least partially overlapping specificities t’oward the peptides generated during carbon-starvation. In addition. t’he results with the pepD+ strain suggest that a significant fraction ( - 359,;) of the peptides that accumulate in the multiple mutant are dipeptides. (c) Protein
synthesis
during
starvation
During starvation no net protein synthesis is possible but amino acid incorporation can occur as a result of protein turnover (Mandelstam, 1958a,b). Amino acids for the synthesis of new proteins are provided by protein degradation. Since the production of amino acids during starvation is sharply reduced in the peptidasedeficient strains, we expected that) the rate of protein synthesis during starvation would also be reduced in the peptidase mutants. To test this expectation, we measured the rate of protein synthesis during starvation for carbon in peptidasedeficient and wild-type strains (Fig. 6). Clearly the simultaneous absence of peptidases N, A. Band D (TN843) leads to a significant decrease in the ability of the cell to s.vnthesize protein during starvation. (d) Growth
charactrristks
of peptidase
mutants
Willets (1967) showed that in E. coli the transfer ofcells growing in a rich medium to minimal medium leads to an immediate increase in protein degradation. This increased degradation presumably provides amino acids for the synthesis of new prot,eins required for the resumption of growth. Since the peptidase-deficient strains show diminished abilities to degrade and synthesize protein during carbon starvation, they might be expected to show similar deficiencies in a shift-down and. as a result, they might have prolonged lag times after such a shift. Figure 7 shows growth curves for the wild-type and a multiply peptidase-deficient mutant. When the cells were shifted from nutrient broth to minimal glucose medium the wild-type showed a lag time of about 60 minutes before resuming exponential growth. The pepNABD strain, TN843. requires more than eight hours for resumpt,ion of growth. (Downshifted cultures of TN843 grow up after overnight, (- 15 h) incubation.) When the broth-grown cells were resuspended in minimal medium supplemented with the 20 r,-amino acids (at 1 x 10m4 M), no lag was observed in LT2. The mutant strain’s lag period was reduced to -30 minutes, Strains containing only one of the four peptidases missing in the pepN pepA pepB pepD strain show lag times intermediate between the mutant and the wild type (Table 3). These results strongly indicate that the observed increase in lag time is caused by the diminished availability of amino acids which results from the combined loss of peptidases N\‘. A, B and D.
(‘.
30
YES,
I,
GREES
I
2
AND
(‘.
(:.
MILLER
4
3
5
Time (h)
kc:. TX873 TX894 TN843
6. Protein synthesis during rarbon~sourw (pepAr+ prp-4 - ppH- pepD-): -U-E+> (prpS- pep.4 - PP~H- p~pD+): -O-o--. (p+Vprpd - pel-‘R- prpI)-).
Doubling
times
starvation. -b-n-, LT:! (wild-type): TN871 (f~irp,.L’~pcpA+ prpB- pepD-); TN875 @p-V- fqut p~pR+ pq~D-);
nn.d lag times of wild-typ
and
mutant
-A-A-, -m-m--. [email protected].
straitts Doubling timr: (min)
LT2 TN843 TN873 TN874 TN8i5 TN894
+ + -
+ + -
+ + -
+ +
70 >4HO 100 70 90 100
56 x0 TO 62 70
70
t Measured after shift from nutrient brot,h to minimal ghwose medium. These times can be reproduced to about _+IS”,. The lag times of the strains with 3 peptidase mutations may not differ significantly from Lx?. 1 In minimal glucose medium.
Doubling times for a series of strains containing various peptidase mutations have also been determined (Table 3). Multiple peptidase deficiency leads to a decreased exponential growth rate as well as prolonged lag times. This observation suggests that these enzymes play some role during exponential growth as well as during starvation.
l’K.OTEIN
DE(:KAD.ATIOS
0.4 -
IN
S/l LXO~VELLrZ
hll’T.AN’l’S
3I
(a)
0.3
-
0.2
-
O-l 008
-
0.06
-
0.04
. I
0.02 -
.
l
lb
-
.
.
.
-
E c z ;; E : G 51
0.01
-
o-5 04
-
I
I
240
300
I
I
I
60
120
1
I
(b)
0.3 o-2
0.1 0.08 0.06 0.04 7
2
0’02
I
180
360
420
Time (min)
PK:. 7. Growth after nutritional shift-down. Ckils growing exponentially in nutrient broth wcw washed and resuspended in minimal glucose medium (a) or minimal glucose medium containing the 20 standard I.-amino acids, each at 0.1 mM (h). -@-a--. LT2 (wild-type); -A-A-, TN843 (prp,V pe~1.1~
pepK prpr). 4. Discussion Each of the peptidases missing in the mutant strains studied in this paper can function in the catabolism of exogenously supplied peptides (Miller & MacKinnon. 1974: Miller & Schwartz, 1978; Kirsh et al., 1978). The major question we set out to answer was : do these peptidases also act in the degradation of intracellular protein during starvation 2 The results presented here indicate that they do function in carbon-starvation-induced protein degradation because in their absence (1) the extent of such degradation is decreased, and (2) the products of degradation are different The production ofpeptides by the starved mutant rather than free amino acids
3’2
('. YEN.
L. GREEK
AND
('. (:. MILLER
as in the wild type, indicates that these enzymes lie on the starvation degradation pathway. The most obvious hypothesis is that these enzymes function late in the degradation pathway, hydrolyzing small (trichloroacetic acid-soluble) peptides produced by the attack of other enzymes on larger polypeptides. If this is true. it is not immediately obvious why the rate of production of acid-soluble material is reduced in the mutant. Vl’e have considered three explanations for this observation. (1) Some of the polypeptides normally attacked by the peptidases missing in the mutant are acid-insoluble. These polypeptides might be large fragments produced by other hydrolytic enzymes or they might be native proteins that are normally degraded uia initial N-terminal aminopeptidase attack. In preliminary experiments employing gel filtration (Sephadex G75 in sodium dodecyl sulfate-cont’aining buffers) of sodium dodecyl sulfate-solubilized starved cells we found no evidence for the presence of a significant amount of labelled material of a size expected for peptides slightly too large to be acid-soluble in either the mutant or the wild-type. If the reduction in the amount of acid-soluble material observed in the mutant resulted from accumulation of material in a pool of peptides slightly larger than those detected as acid-soluble. we would expect as much as 15 t)o 20% of the total protein label to appear in this pool. (2) A second possible explanation for the observed reduction is that the peptides themselves might directly inhibit the action of other peptidases acting earlier in the degradation pathway. We know that the most deficient mutant studied here still contains several peptidase activities (Miller $ MacKinnon, 1974: McHugh & Miller, 1974: Yaron et al., 1972). At least, one of the peptidases still present in our mutant strain is known to be inhibited by di- and tripeptides (Yaron. 1974). (3) It is also possible that the reduction in the extent of formation of acid-soluble protein fragment,s during carbon-starvation is an indirect result of peptidase loss. Several lines of evidence indicate that protein degradation requires metabolic energy. St clohn & (Goldberg (1978) have proposed that in E. coli the intracellular ATT’ concentration must remain above a minimal level for protein turnover to occur. If metabolism of the amino acids produced by turnover makes a significant contribution to the energy supply of the cell during carbon-starvation, the failure of the mutants to produce free amino acids at the wild-type rate might result in a decrease in cellular ATP concentration. As a result, the initial steps of protein breakdown might not take place at the normal rate. The data available do not allow us to distinguish among these possibilities. The results of the turnover studies on strains containing only one of the four missing peptidases indicate that these peptidases show some overlap in their specificities toward peptides generated internally just as they do toward those supplied exogenously. Studies of peptide utilization by the strains having only one of the four peptidases have indicated that most small peptides can be hydrolyzed efficiently by more than one of these enzymes (Kirsh et al., 1978; Green, unpublished results). The large increase in the production of free leucine in these strains (particularly pepN+ or pepA+) compared to the quadruple mutant suggests that many of the peptides generated during turnover can be hydrolyzed by more than one of the missing peptidases. In the absence of a group of peptidases, protein turnover during starvation is blocked at a late step in which small peptides are usually converted to free amino
PROTEIS
DEGRADATION
IN
SA 1,:1fO.\‘\‘ELLd
Ml’TAX’I’S
33
acids. As a result of this block, starving mutant cells cannot synthesize protein at’ wild-type rates and growth lag times after a nutritional shift are greatly prolonged. These results support Mandelstam’s (1963) proposal that protein turnover during starvation constitutes an important mechanism of adaptation in bacteria. They also establish the importance of peptide-hydrolyzing enzymes in the protein turnover process. This work was supported by grants from the United States Public Health Service. by Research (lareer Development Award GM07321 from the National Institute for General Medical Sciences (to C. M.), and by Research grant AT1 0333 from the National Institute of Allergy and Infectious Disease. REFERENCES Goldberg, A. L. (1972). Proc. Nat. Acad. &i..lT.S.A. 69, 422-426. Goldberg. A. L. 8: St John, A. C. (1976). Annu. Rev. Biochem. 45, 747-803. Heiman. C. & Miller, C. G. (1978). J. Bacterial. 135, 588-594. Kirsh. M.. Dembinski, D. R., Hartman, P. E. &Miller, C. G. (1978). J. Bacterial. 134,36lL374. Kleckner. N., Roth, J. & Botstein, D. (1977). J. ~lilol. Biol. 116, 125159. Mandelstam, J. (1958a). Biochem. J. 69, 1033110. Mandelstam, J. (1958b). B&hem. J. 69, 116119. Mandelstam, J. (1960). Bactertil. Rev. 24, 289-308. Mandelstam, J. (1963). Ann. N.Y. Acud. Sci. 102, 621-636. McHugh, G. L. & Miller, C. G. (1974). J. Bacterial. 120, 364-371. Miller, C. G. (1975a). Annu. Rev. Microbial. 29, 485.504. Miller. C. G. (1975b). J. Bacterial. 122, 171-176. Miller. C. (:. & MacKinnon, K. (1974). J. Bacterial. 120, 355363. Miller, C. G. & Schwartz, G. (1978). J. Bucteriol. 135, 6033611. Miller, C. (:. 62 Zipser, D. (1977). J. Bactrriol. 130, 347-353. Miller, (1. G., Heiman, C. & Yen, C. (1976). J. Bacterial. 127, 490-497. Patterson, &I. S. & Greene, R. C. (1965). Anal. Chem. 37, 854-857. Pine, M. J. (1972). Annu. Rev. Microbial. 26, 103-126. Roth, ,J. R. (1970). In Methods of Enzymology (Tabor. H. & Tabor. C. W.. eds). vol. 17a, pp. 2235, Academic Press Inc., New York. Schlrssinger, D. & Ben-Hamida, F. (3966). Biochim. Biophys. Actu, 119, 171L182. St John, A. C. 8: Goldberg, A. L. (1978). J. Biol. Chem. 253, 27052711. Yogt, V. M., Eisenman, R. & Diggelmann, H. (1975). J. Mol. Riol. 96, 471-493. Willets, N. S. (1967). Biochem. J. 103, 4533461. Yaron. A. (1974). Israel J. Chem. 12, 651-662. Yaron. A., Mlynar, D. & Berger, A. (1972). Biochem. Biophy. Res. (‘ommun. 47, 897-902.