Escherichia coli contains a soluble ATP-dependent protease (Ti ...

Proc. Nadl. Acad. Sci. USA Vol. 84, pp. 5550-5554, August 1987 Biochemistry

Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La BYUNG JOON HWANG*, WOO JIN PARK*, CHIN HA CHUNG*t, AND ALFRED L. GOLDBERGt *Department of Zoology, College of Natural Science, Seoul National University, Seoul 151, Medical School, Boston, MA 02115

Korea; and tDepartment of Physiology and Biophysics, Harvard

Communicated by Bernard D. Davis, April 13, 1987 (received for review January 20, 1987)

on glycerol gradient centrifugation (8, 21), we concluded previously that this activity was due to a defective form of protease La that was more labile than the wild-type enzyme (8, 21). However, these studies did not test critically the alternative possibility that the residual ATP-dependent activity is due to a distinct enzyme. One problem complicating interpretation of these studies was uncertainty as to whether these ion mutants retain some protease La function. Maurizi et al. (5) showed that cells completely lacking protease La (because of a TnlO insertion in the Ion gene) are viable, have only a partial defect in energy-dependent degradation of abnormal polypeptides, and show no reduction in proteolysis upon nitrogen starvation (5). These authors concluded that E. coli must contain an additional ATP-dependent proteolytic enzyme (5). The present studies were undertaken to critically examine this suggestion and to determine whether the ATP-stimulated proteolytic activity that we found in the extracts of ion mutants is in fact a labile form of protease La, as concluded previously (8, 21), or whether it represents a distinct enzyme. Recently, Katayama-Fujimura et al. (22) obtained direct evidence for a new soluble ATP-requiring protease in such cells, and a report on their findings has appeared since our studies were submitted for publication. In this communication we also demonstrate that E. coli contains an ATPdependent proteolytic enzyme differing from protease La in many important features.

The energy requirement for protein breakABSTRACT down in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the Ion gene product. We have partially purified another ATP-dependent protease from loncells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [3H]methyl-casein to acid-soluble products in the presence of ATP and Mg2+. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. We propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show proteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of KatayamaFujimura et al. [Katayama-Fujimura, Y., Gottesman, S. & Maurizi, M. R. (1987) J. Biol. Chem. 262, 4477-4485] indicate that E. coU contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles. In Escherichia coli, as in eukaryotic cells, metabolic energy is required for both the rapid degradation of highly abnormal polypeptides (1-3) and the increased breakdown of normal cell proteins during starvation (3, 4). Physiological and genetic studies indicate that these two degradative processes involve distinct energy-dependent proteolytic systems (1, 5-7). A critical enzyme in the hydrolysis of abnormal proteins in E. coli is the ATP-dependent endoprotease La, which is the product of the Ion gene (also called deg or capR) (2, 5, 8, 9). This enzyme degrades proteins and ATP by a coupled mechanism (10-12) in which two ATP molecules are consumed per peptide bond cleaved (12). Mutants in the ion gene have a reduced capacity to hydrolyze various types of abnormal polypeptides (e.g., nonsense fragments or analogcontaining proteins) (2, 5, 8, 13) and certain critical regulatory proteins (14, 15). However, in ion- cells such proteins are still degraded relatively quickly compared to the bulk of cell proteins (5, 8, 16), and this process also requires ATP (5). Although a number of endoproteases have been isolated from E. coli (2, 17), the enzymes that catalyze this Ionindependent protein breakdown in vivo are unknown. Extracts of Ion mutants show proteolytic activity that is stimulated by ATP in both soluble (18) and particulate (19-20) fractions. Because this soluble activity resembled protease La in its behavior on DEAE-cellulose chromatography and

MATERIALS AND METHODS Bacterial Strains. The isogenic E. coli strains RGC121 (ion+), RGC121/pJMC40 (lon'), RGC123 (Ion-), and RGC125 (Ion-) were originally provided by A. Markovitz (University of Chicago). RGC121/pJMC40 carries the wildtype Ion' allele both on the chromosome and on a recombinant plasmid (23). RGC123 carries the IonR9 allele (21, 23-25), and RGC125 contains a Ion- allele used in our prior studies (8, 21) and previously referred to only as R-. Materials. Phosphocellulose and DEAE-cellulose were purchased from Whatman, Sephacryl S-300 from Pharmacia, nitrocellulose papers (0.45 Aum pore size) from Schleicher & Schuell, [3H]formaldehyde from New England Nuclear, and _25I-labeled protein A from Amersham. All other reagents were obtained from Sigma. [C3H3]Casein was prepared as described (8, 17). Purification of the ion Gene Product. Protease La was purified from RGC121/pJMC40, and the R9 protein (lonR9 product) from RGC123, as described (17, 21). These proteins were more than 95% pure as determined by NaDodSO4/ polyacrylamide gel electrophoresis (26). Immunochemical Detection of Protease La. Exponentially growing cells were harvested, resuspended in 2% (wt/vol) NaDodSO4, and electrophoresed in 10% (wt/vol) polyacryl-

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Abbreviations: iPrP-F, diisopropyl fluorophosphate; MalNEt, N-

ethylmaleimide.

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Biochemistry: Hwang et al. amide slab gels (26). After electrophoresis, the proteins were transferred to nitrocellulose paper at 45 V for 90 min in a "transblot" apparatus assembled as described by Stott et al. (27). The papers were incubated in 5% (wt/vol) bovine serum albumin for 2 hr at room temperature and then in the antibody solution at 40C overnight. They were washed with 5 mM Tris/HCl buffer (pH 7.4) containing 200 mM NaCl and 0.1% (vol/vol) Triton X-100 and were incubated in 125I-labeled protein A solution (200,000 cpm/ml). After incubation, the nitrocellulose papers were washed three times with this same buffer, dried, and exposed overnight to Kodak X-Omat R film for autoradiography (28). The antibody against protease La was prepared by injecting 0.5 mg of the purified protease emulsified with complete Freund's adjuvant. Two weeks later, the rabbits received the first of three booster injections consisting of 0.25 mg of the enzyme in incomplete adjuvant administered at weekly intervals. Rabbits were then bled by heart puncture, and the IgG fraction was isolated by ammonium sulfate fractionation and DEAE-cellulose chromatography (29). Enzyme Assays. Proteolytic activity in the presence and absence of 2 mM ATP was measured using [C3H3]casein as the substrate. The production of radioactive material soluble in trichloracetic acid was measured as described (8, 17). A typical reaction mixture contained 50 mM Tris/HCl (pH 8), 5 mM MgCI2, 1 mM dithiothreitol, 0.5 mM EDTA, and 10 jig of casein in a total volume of 0.1 ml. DEAE-Cellulose Chromatography of Cell Extracts. The lon- strain RGC125 was grown to stationary phase at 37°C in Luria broth and harvested. All subsequent work was carried out at 4°C. The cell paste (90 g) was resuspended in 200 ml of 10 mM Tris/I-CI buffer (pH 7.8) containing 10 mM 2mercaptoethanol, 5 mM MgCl2, 1 mM EDTA, and 10% (vol/vol) glycerol. The cells were then disrupted with a French press at 14,000 psi (1 psi = 6.89 kPa) and centrifuged at 100,000 x g for 2 hr. The supernatant, containing 11 g of protein, was loaded on a DEAE-cellulose column (5 x 25 cm) that had been equilibrated with the buffer. After the column was washed extensively, proteins were eluted with a lihear gradient (4 liters) of 0-0.2 M NaCl at a flow rate of 200 ml/hr; fractions of 20 ml were collected. Gel Filtration. The sizes of protease Ti and its two components were estimated by gel filtration. The protease obtained from the DEAE-cellulose and the flow-through (P) and bound (A) components from the phosphocellulose column were loaded on a Sephacryl S-300 column (1.1 x 28 cm) that had been equilibrated with 20 mM Tris/HCl buffer (pH 8) containing 2 mM 2-mercaptoethanol, 5 mM MgCI2, 1 mM EDTA, and 10% glycerol. Fractions of 0.45 ml were collected at a rate of 5 ml per hr. When either component A or P (see Table 1) was fractionated on the column, its activity was assayed- by incubating the eluted fractions with the other component (unfractionated). Marker proteins used were thyroglobulin (669 kDa), apoferritin (443 kDa), catalase (240 kDa), alcohol dehydrogenase (150 kDa), and hexokinase (100 kDa).

Proc. Natl. Acad. Sci. USA 84 (1987) b

a

c

A Second Soluble ATP-Dependent Protease in E. coli. In prior studies (8), we partially purified an ATP-requiring

proteolytic activity from lon- strains RGC125 and RGC123. This casein-degrading activity was eluted from DEAEcellulose columns and sedimented in glycerol gradients in a similar fashion to protease La, but it lost activity upon phosphocellulose chromatography or upon exposure to high salt concentrations (8, 21). To examine whether this activity represents an altered form of La or is actually a distinct enzyme, we performed immunoblotting of these extracts with an antibody against the purified Ion gene product (Fig. 1).

d

87 kDa- FIG. 1. Immunochemical detection of protease La in isogenic lon+ and lon- cells. Samples (1 ml) of cells in logarithmic phase were treated as described in Materials and Methods. Lanes: a, 0.1 ,g of purified protease La; b, wild-type cell (lon'); c, lonR9 (RGC123); d, lon- (RGC125). In prior studies, this band was identified as 92-94 kDa based on electrophoretic mobility, but in fact it is an 87-kDa polypeptide, based on its amino acid sequence (D. Chin, S. A. Goff, and A.L.G., unpublished data).

Both the wild-type extract and the extract of RGC123, which carries the lonR9 allele, contained an immunoreactive polypeptide indistinguishable in size from the purified wild-type subunit (87 kDa). The R9 polypeptide corresponds to an inactive subunit of protease La that was shown to inhibit the wild-type enzyme by forming mixed tetramers (2, 24). In contrast, the lon- strain RGC125 lacked any polypeptide that crossreacted with the antibody against protease La. Nevertheless, when the extract of this mutant was chromatographed on DEAE-cellulose (Fig. 2), a single peak of ATPdependent proteolytic activity was found, as reported previously (8). When the active fractions were pooled, dialyzed, and concentrated, ATP (2 mM) was found to stimulate 15-fold the hydrolysis of [C3H3]casein (Fig. 3) and of ['4C]globin. Casein degradation was linear for at least 2 hr and was proportional to protein concentration (data not shown). E. coli thus contains at least two soluble ATP-dependent proteases. We propose to name the second activity "protease Ti" to distinguish it from the other soluble casein-degrading enzymes (2, 17) in these cells, proteases Do, Re, Mi, Fa, So, and La. Dissociation of Protease Ti into Distinct Components. To purify protease Ti further, the active fractions from the DEAE-cellulose column were dialyzed against phosphate buffer containing 10% (vol/vol) glycerol (Table 1) and then were loaded on a phosphocellulose column. Most proteins failed to bind to this column. Neither the flow-through (P) nor the bound (A) fraction (eluted with buffer containing 0.3 M phosphate) showed any ATP-dependent casein degradation by itself. However, when these two inactive fractions were mixed together, more than 90% of the original proteolytic activity was reconstituted (Table 1). Thus, protease Ti P 8B

A

16 .-

0

14

0 I O

OD NM

Un

z

RESULTS

5551

_E)

12 -, z 10

'I) 08

FRACTION NUMBER (x2Oml)

FIG. 2. Elution profile of the ATP-dependent proteolytic activity. Extract of E. coli RGC125 (Ion-) was subjected to DEAE-cellulose chromatography, and casein-degrading activity in every third fraction was assayed in the presence (o) or absence (o) of 2 mM ATP. Solid bars indicate where the flow-through (A) and bound (B) fractions from the phosphocellulose column (see Table 1) were eluted on DEAE chromatography. ATP-dependent casein hydrolysis was observed in these fractions when 20 ,ul was incubated with 5 ,ul of the complementary (A or B) fraction from the phosphocellulose column.

5552 0

Biochemistry: Hwang et al.

15

-

o 100 z

w

5-

(n

u

OL

I5

30 45 TIME (min)

60

FIG. 3. ATP-dependent casein hydrolysis by protease Ti. The active fractions (400 ml) obtained from the DEAE-cellulose column were pooled and concentrated to a final volume of 18 ml by ultrafiltration (Amicon PM10 membrane). Aliquots (5 ,l) of the sample were then incubated for different periods in the presence (e) or absence (o) of ATP (2 mM).

contains at least two different components, neither of which has proteolytic activity by itself. Because protease Ti dissociates in 0.1 M phosphate buffer into two components separable on the phosphocellulose column (Table 1), it appeared possible that on DEAEcellulose chromatography these two components were also separated partially, so that the ATP-dependent proteolytic peak in Fig. 1 represents the fractions where both components were present (i.e., where the peaks of the two components overlapped). To test this possibility, aliquots of the flow-through fraction (P) or of the bound fraction (A) from the phosphocellulose column (Table 1) were added to the individual fractions from the DEAE-cellulose column, and ATPdependent proteolysis was measured. As indicated by the solid bars in Fig. 2, the two components must have been eluted from the DEAE column at different salt concentrations, since addition of A or P generated ATP-dependent proteolytic activity in previously inactive fractions in different regions of the columns. Both components were present in those fractions where ATP-dependent proteolysis was initially detected (without reconstitution). Thus, the different subunits appear to be held together by ionic interactions, and the proteolytic peak shown in Fig. 1 represents only a portion of the total protease Ti activity in these cells. To determine the sizes of protease Ti and its two components, each was subjected to gel filtration on a Sephacryl S-300 column under nondenaturing conditions. The ATPdependent proteolytic activity obtained either from the DEAE-cellulose column (Fig. 2) or by reconstitution of the fractions from phosphocellulose chromatography (Table 1) was eluted as a sharp peak corresponding to 370 kDa (data not shown). On this same column, the fraction that did not bind to phosphocellulose was eluted with an apparent molecular mass of 260 kDa and the bound one at 140 kDa in low-salt buffers (data not shown). Therefore, protease Ti consists of at least two essential components of different sizes, in Table 1. Separation of components of protease Ti by phosphocellulose chromatography and their reconstitution Casein hydrolysis, % Fraction ATP + ATP 1.5 2.1 Flow-through (P) Bound (A) 0 0.4 1.8 30.4 Flow-through plus bound Protease Ti obtained from the DEAE-cellulose column was concentrated and dialyzed against 0.1 M K2HPO4 buffer (pH 6.5) containing 2 mM 2-mercaptoethanol, 5mM MgCI2, 1 mM EDTA, and 10% glycerol. The proteins (180 mg) were then loaded on a phosphocellulose column (2.5 x 10 cm) equilibrated with the same buffer. After the flow-through fractions were collected, the proteins that bound to the column were eluted with the same buffer containing 0.3 M phosphate. The two fractions were concentrated by ultrafiltration to the original volume and dialyzed against 20 mM Tris/HCI buffer (pH 8) containing 2 mM 2-mercaptoethanol, 5 mM MgCI2, 1 mM EDTA, and 10% glycerol. [C3H3]Casein hydrolysis by each fraction and by the mixture (flow-through plus bound) was then measured in the presence or absence of ATP.

Proc. Natl. Acad. Sci. USA 84 (1987)

La, which is a tetramer of identical 87 kDa subunits (17). These subunits also differ in their stability and catalytic properties. Upon incubation at 370C by itself, the bound fraction (A) lost almost 97% of its activity in 1 hr, while the flow-through (P) appeared completely stable (Table 2). The presence of ATP (2 mM) during this preincubation completely stabilized the bound component. In addition, ATP also partially protected this subunit of the enzyme against inactivation by MalNEt, a thiol-blocking agent (see below). Therefore, we refer to it as component A, since it must bind ATP. Related studies demonstrated that this fraction, unlike the flow-through material, has appreciable ATPase activity, although it lacked proteolytic activity (Table 1). In a typical incubation, this fraction consumed in 1 hr about 17% of the ATP added (2 mM). This ATPase activity copurifies with the ability to restore ATP-dependent proteolytis (data not shown). Further, the ATPase activity of the bound component (A) was not increased by addition of the other component (P). This protease is also inhibited by iPr2P-F, which specifically inactivates serine proteases (see below). When each of these components was incubated with iPr2P-F (20 mM) to modify serine residues in the proteolytic site, only the bound fraction was susceptible to this covalent inhibitor. Since this fraction contains the proteolytic active site, we refer to it as component P. Cofactors and Inhibitors of Protease Ti. A variety of nucleotides were tested for their ability to support protease Ti activity (Table 3). With the exception of dATP, no other nucleoside triphosphate tested (including CTP, UTP, or GRP) could substitute for ATP. Thus, this enzyme has stricter nucleotide specificity than protease La (8, 10, 30). AMP had no effect, while ADP supported activity about 20% as well as ATP, although in these partially purified preparations, there may be some conversion of the ADP to ATP (18). Because the nonhydrolyzable ATP analogs p[CH2IppA and p[NH]ppA also cannot substitute for ATP, proteolysis seems to require concomitant ATP hydrolysis, as was found for protease La (8, 10-12). To further characterize protease Ti, several site-directed inhibitors and inhibitors of protease La were tested against it. Protease Ti seems to be a serine protease, since it is strongly inhibited by iPr2P-F (Tables 2 and 4). In addition, protease Ti appears to require free sulfhydryl group(s) for function. contrast to protease

Table 2. Differential inactivation of the two components of protease Ti upon ATP removal and by diisopropyl fluorophosphate treatment % activity remaining after incubation at 37°C Flow-through Bound Preincubation fraction (P) fraction (A) With ATP 100 100 Without ATP 103 7 With MalNEt 116 1 With MalNEt and ATP 127 22 With iPr2P-F and ATP 7 98 The two fractions obtained from the phosphocellulose column (Table 1) were subjected to gel filtration (see Materials and Methods) and then incubated with or without ATP (2 mM) for 1 hr at 37°C. The incubations with diisopropyl fluorophosphate (iPr2P-F, 20 mM) or N-ethylmaleimide (MaINEt, 2 mM) were for 30 min. After dialysis for 8 hr with buffer changes every 2 hr, the ability of these preparations to support proteolysis in the presence or absence of ATP was assayed by mixing with an equal volume of the complementary unincubated material. In the absence of ATP, iPr2P-F caused a similar inhibition of the flow-through fraction and caused no further reduction in the activity of the bound fraction.

Biochemistry: Hwang et al. Table 3. Effects of various nucleotides on casein hydrolysis by proteases Ti and La Relative activity, % Nucleotide Ti La ATP 100 100 dATP 76 120 CTP 1 104 UTP 0 89 GTP 0 11 ADP 21 3 AMP 1 2 0 0 p[CH2]ppA p[NH]ppA 0 4 Protease Ti (obtained by DEAE-cellulose chromatography) and purified protease La were assayed in the presence of various nucleotides (2 mM). The basal activity seen without any nucleotide added (-8% of that with ATP) was subtracted from all values. p[CH2]ppA, adenosine 5'-[P,y-methyleneltriphosphate; p[NH]ppA, adenosine 5'-[,3,y-imido]triphosphate.

MalNEt and p-chloromercuribenzoate were strongly inhibitory, although iodoacetamide had no effect. The essential SH residue(s) are located on component A, which contains the ATPase activity (Table 2). Further, this enzyme was stimulated -2-fold by dithiothreitol and by EDTA, which together were additive in enhancing proteolysis. However, it is not sensitive to inhibition by leupeptin (31) or Ep475 (32), which specifically inactivate proteases with thiol residues in their active sites. Little or no inhibition of protease Ti was observed with any concentration of the lonR9 gene product (Fig. 4), which inhibits protease La by forming mixed tetramers (2, 24). At low concentrations, vanadate inhibits protease La almost completely by preventing ATP binding (33) and inhibits the ATP-dependent proteolysis in mitochondria (34) and reticulocytes (35). However, vanadate (0.1 ,uM) had no inhibitory effect on these preparations of protease Ti (and caused only a slight inhibition of the homogeneous enzyme).Thus, proteases La and Ti differ in many properties.

DISCUSSION These studies have demonstrated a second type of soluble ATP-dependent protease in E. coli. This multicomponent Table 4. Effects of various protease inhibitors on ATP-dependent casein hydrolysis by protease Ti Addition(s) Relative activity, % None 100 iPr2P-F (1 mM) 101 iPr2P-F (10 mM) 6 o-Phenanthroline (1 mM) 81 EDTA (1 mM) 227 Dithiothreitol (1 mM) 176 EDTA plus dithiothreitol (both 1 mM) 315 MaINEt (1 mM) 44 MalNEt (5 mM) 26 p-Chloromercuribenzoate (0.1 mM) 1 lodoacetamide (5 mM) 99 Leupeptin (50 ,ug/ml) 111 Ep475 (50 ,g/ml) 101 Protease Ti (obtained by DEAE-cellulose chromatography) was dialyzed extensively against 10 mM Tris/HCI buffer (pH 8) containing 5 mM MgCI2 and 10% glycerol. Reaction mixtures (0.1 ml) containing 50 mM Tris/HCI (pH 8), 5 mM MgCI2, and the indicated inhibitors were incubated for 10 min at 37°C prior to the addition of casein. The low activity seen in the absence of ATP ('8% of that with ATP) was subtracted from all values.

Proc. Natl. Acad. Sci. USA 84 (1987)

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2. 100

,s, 50

LLJ cr

C

1.5 R9 PROTEIN (og)

3

0.2 0.1 VANADATE (IM)

FIG. 4. Effects of the lonR9 gene product, vanadate, and heparin

on casein hydrolysis by protease Ti (o) and purified protease La (o). Protease Ti was prepared as for Fig. 3. The low amount of activity (6%) without ATP was subtracted from that with 2 mM ATP present. Protease La showed no activity in the absence of ATP. These

inhibitors were added to reaction volumes of 0.1 ml.

enzyme is evidently identical to that reported recently by Katayama-Fujimura et al. (22) in another lon- strain lacking protease La and in wild-type cells. In addition to protease La and the second enzyme, which we call protease Ti, there exists in these cells an ATP-stimulated proteolytic activity tightly associated with the cell membrane (19, 20). This activity is present in strains lacking protease La (20) and differs from both Ti and La in substrate preference. E. coli also contains five soluble casein-degrading proteases (17), named Do, Re, Mi, Fa, and So, that can function independently of ATP. Although they differ in size and net charge from protease Ti, it is possible that one or more of these enzymes shares a subunit with or is derived from either protease Ti or the membrane-linked enzyme. Proteases Ti and La clearly differ from each other in many biochemical properties, including nucleotide specificity, sensitivity to inhibitors, subunit composition, and substrate specificity (22). Nevertheless, they do share certain general characteristics. Both appear to be serine proteases as shown by their sensitivity to iPr2P-F. [Although Katayama-Fujimura et al. (22) did not obtain evidence for this conclusion, they utilized less reactive inhibitors that do not inactivate many serine proteases, including protease La.] Both ATPdependent enzymes also contain essential thiol residues (ref. 22 and Table 2), and both degrade [C3H3]casein or [14C]globin but not 125I-labeled insulin. Moreover, they behave similarly on ion-exchange chromatography and on density-gradient centrifugation (8, 21). In fact, because of these similarities in multimeric size and net charge, we failed previously to recognize that the ATP-dependent peak in these Ion mutants is due to a new enzyme. This residual activity was attributed to a "labile form" of protease La that was easily inactivated by salts and by phosphocellulose chromatography (6, 10). It is now clear that these treatments must have dissociated the subunits of protease Ti (Fig. 1), which interact with each other through ionic bridges. Since protein hydrolysis should not require metabolic energy, there has been much interest in the role of ATP hydrolysis in proteolysis in eukaryotes and prokaryotes (2, 10, 11, 33, 35, 36). The ATP-dependent mechanism of protease Ti seems to differ from that of protease La. The latter enzyme is composed of four identical subunits, each of which contains a nucleotide binding site sensitive to vanadate (33). These subunits function cooperatively in a complex reaction cycle that is specifically activated upon binding of protein substrates (10, 12, 37). In contrast, protease Ti consists of at least two components that differ from each other in size (ref. 22 and Fig. 3), net charge, stability, and function. Although the sizes of the polypeptides comprising these components are still uncertain, their enzymatic roles clearly differ. The component that binds strongly to phosphocellulose (A) and that contains essential cysteine residue(s) is labile in the absence of ATP (Table 2 and ref. 22).

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Biochemistry: Hwang et al.

This fraction by itself shows ATPase activity, which is activated by protein substrates (unpublished observations). The other component (P) contains the active site of the protease that is sensitive to iPr2P-F; yet both components are required for ATP-dependent proteolysis. Protease Ti, like the other cytosolic proteases in E. coli (17) and in mammalian cells (34, 35), is a very large multisubunit complex. In contrast, the majority of well-characterized proteases func-

tion extracellularly and are small enzymes, presumably because they require less stringent regulation than these cytosolic proteases (37). Important questions for future work concern the specificity, the functions, and the relative importance of these different ATP-dependent proteases. It is noteworthy that in the lonstrain studied here, the breakdown of puromycyl and canavanine-containing polypeptides occurs at about 50% of the rate seen in wild-type cells (6). Accordingly, extracts of these cells show an reduction in the activity of the ATP-stimulated 50% proteolytic peak that contains both proteases La and Ti. Protease Ti clearly accounts for this ATP-dependent activity in the Ion- mutant lacking a discernible gene product, but it also accounts for the residual activity in the lonR9 allele (RGC123). Even though the lonR9 gene product forms tetramers like the wild-type protease La (8, 21, 23), the ATP-dependent peak in such cells shows the characteristic features of protease Ti. The actual amount of protease Ti present in these cells is probably underestimated by such analyses using ion-exchange chromatography, because high salt dissociates this enzyme into inactive components (ref. 22 and Fig. 2), one ofwhich is quite labile. At the least, protease Ti seems to be a major cellular proteolytic enzyme, whose total activity can be similar to or greater than that of protease La. However, it is possible that in the Ion- cells studied here, protease Ti is induced to compensate for the loss of protease La. Protease Ti, like protease La, may thus play an important role in the degradation of abnormal proteins. It has recently been shown that Ion is a heat shock gene (2, 16, 38). The intracellular level of protease La (16) and the cell's capacity to degrade abnormal proteins (16, 39) are reduced in htpR (rpoD) mutants. Moreover, when cells produce large amounts of abnormal proteins, transcription of Ion and other heat shock genes rises (40). Recent studies indicate that other heat shock proteins are also important for energy-dependent breakdown of abnormal proteins (refs. 16 and 38; unpublished observations). Possibly, protease Ti is also regulated as part of the heat shock response and may help prevent the accumulation of abnormal proteins in vivo. Alternatively, protease Ti may function in the accelerated breakdown of normal proteins that occurs in bacteria starved for a carbon or nitrogen source (7, 21), certain ions (41) or a required amino acid (4, 7). This response, like the breakdown of a number of short-lived proteins in E. coli (15, 42), appears normal in Ion- cells (1, 5). Definitive information concerning the function(s) of protease Ti in vivo clearly must await the isolation of mutants lacking this enzyme. We are grateful to Dr. Maurizi for the suggestion of a multicomponent protease and to him and Dr. Gottesman for communicating findings prior to publication. We thank Ms. Eun Young Lee and Mrs. Aurora Scott for their help in the preparation of this manuscript and Mr. Ethan Benardete and Thomas Moll for assistance in certain experiments. This work was supported by research grants from the Ministry of Education and the Science and Engineering Foundation

of Korea and the National Institute of Neurological Disease and Stroke. 1. Goldberg, A. L. & St. John, A. C. (1976) Annu. Rev. Biochem. 45, 747-803. 2. Goldberg, A. L. & Goff, S. A. (1986) in Maximizing Gene Expression, eds. Reznikoff, W. & Gold, L. (Butterworths,

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