THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 268, No. 11, Issue of April 15, pp. 8193-8198, 1993 Printed in U. S.A.

Membrane Vesicles Containing Overproduced SecY and SecE Exhibit High Translocation ATPase Activity and Countermovement of Protons in a SecA- and Presecretory Protein-dependent Manner*

(Received for publication, July 27, 1992)

Satoko Kawasaki, Shoji Mizushima, and Hajime Tokuda$ From the Institute of Applied Microbiology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan

Everted membrane vesicles were prepared from Escherichia coli cells containing either overproduced amounts (OP-membrane vesicles) or normal amounts (normal membrane vesicles) of SecY and SecE, both of which are essential components of the protein trans- location apparatus. The rates of translocation of pro- OmpA were similar in the two types of membrane vesicles, whereas translocation ATPase activity, which requires SecA, a precursor protein (pro-OmpA), and membrane vesicles, was appreciably higher with OP- membrane vesicles than with normal membrane vesi- cles. Since ATP hydrolysis has been shown to take place at an earlier part of the translocation reaction, these results suggest that the overproduction of SecY and SecE enhanced the activity of the earlier process, but not the entire process, of the translocation reaction. The addition of pro-OmpA in the presence of SecA caused the partial collapse of ApH (inside acidic) gen- erated on OP-membrane vesicles, suggesting that pro- tons come out from the inside of the membrane vesicles in a pro-OmpA-dependent manner. The collapse of ApH caused by pro-OmpA required SecA, ATP, and SecY and was not detected when normal membrane vesicles were used. These results indicate that the early event of protein translocation, which requires the function- ing of SecA, SecY, and SecE, causes the countermove- ment of protons.

Translocation of presecretory proteins across the cyto- plasmic membrane of Escherichia coli takes place via a specific apparatus composed of several Sec proteins (1). SecA, a peripheral membrane protein, is essential for the initiation of protein translocation and exhibits ATPase activity (2). SecD, SecE, SecF, and SecY are integral membrane proteins (3-6) and are thought to form the apparatus that conducts secretory proteins. Interaction between SecA and SecY or SecE has been reported (7-9). SecE, SecY, and SecA have been sepa- rately purified to homogeneity and used to reconstitute a protein translocation apparatus (10). Success in the reconsti- tution of a translocation apparatus from the three purified Sec proteins has established that these Sec proteins are di- rectly involved in protein translocation. The roles of SecD and SecF in protein translocation, on the other hand, have not been clarified in the reconstitution system.

Protein translocation in E. coli requires two types of energy

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 To whom correspondence should be addressed. Tel.: 3-3812-2111 (ext. 7832); Fax: 3-3818-9435.

source, ATP and a proton motive force (11). The mechanisms whereby the translocation apparatus utilizes these energy sources for protein translocation remain largely unknown. ATP is absolutely required for the initiation of protein trans- location, whereas the requirement for the proton motive force is not always absolute (11). An intramolecular loop formed through disulfide bonding near the carboxyl-terminal region of pro-OmpA, a precursor of major outer membrane protein OmpA, made the translocation of pro-OmpA absolutely de- pendent on the proton motive force (12). These results taken together indicate that ATP and the proton motive force play different roles in protein translocation. These results also suggest that the structure of the translocation apparatus changes upon the generation of the proton motive force so as to conduct secretory proteins, being otherwise incompetent as to translocation.

The proton motive force has two components: an electrical component, membrane potential (Aq),’ and a chemical com- ponent, ApH, both of which stimulate protein translocation. A* (positive at the periplasmic side) may facilitate the elec- trophoretic movement of the negatively charged portions of secretory proteins toward the periplasm, and ApH (acidic at the periplasmic side) may facilitate the translocation of basic amino acid residues by stimulating deprotonation before translocation and protonation after translocation. However, the translocation of a secretory protein that has no charged amino acid residues in its mature portion was found to be significantly stimulated upon the generation of the proton motive force (13). Therefore, the above mechanisms, if any, are not the main reason for the stimulation of protein trans- location by the proton motive force. It is reasonable to assume that the proton motive force directly energizes the apparatus. Possible protein/proton antiporter activity has been discussed as a function of the translocation apparatus (14).

The requirement of the proton motive force is characteristic of the bacterial apparatus, whereas no requirement is known for protein translocation across the membrane of the endo- plasmic reticulum (ER). By means of an electrophysiological technique, the ER apparatus was reported to have a protein conducting channel, which opened upon the discharge of nascent proteins and conducted ions nonspecifically (15). By using the same technique, the signal peptide was shown to cause an increase in ion flux across the membrane of E. coli (16). The involvement of Sec proteins in the increase in ion flux was not examined, however. It was recently reported that protein translocation makes the E. coli membrane permeable to halide anions (17). Since protein translocation does not

The abbreviations used are: A*, membrane potential; ER, endo- plasmic reticulum; IPTG, isopropyl-l-thio-0-D-galactopyranoside; DCCD, N,N”dicyclohexylcarbodiimide; CCCP, carbonyl cyanide m- chlorophenylhydrazone; PAGE, polyacrylamide gel electrophoresis.

8193

8194 Translocation ATPase and Sec Apparatus-dependent H“ Flux

require halide anions (17), the physiological significance of this phenomenon is unclear.

We show in this paper that membrane vesicles containing overproduced SecY and SecE, both of which play fundamental roles in the translocation apparatus, exhibit the SecA- and pro-OmpA-dependent countermovement of protons, which seems to be coupled to the early stage of protein translocation.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids-E. coli W3110 M25 (ompT) (18) was transformed with both pMAN809 and pMAN510 for the simul- taneous overproduction of SecY and SecE (19). The overproduction was induced at 37 “C by the addition of isopropyl-P-D-thiogalactopyr- anoside (IPTG) as reported (19). Cells were harvested at a specified time after the induction of overproduction. Plasmid pSIO53 (20) carries the ompA gene that codes for outer membrane protein OmpA.

Preparation of Everted Membrane Vesicles-Everted membrane vesicles were prepared from either E. coli W3110 M25 cells or W3110 M25 cells harboring both pMAN809 and pMAN510, a SecY/SecE overproducer, as reported previously (20). The amount of membrane vesicles was expressed as that of protein, which was determined by the method of Lowry et al. (21).

Preparation of SecA, pro-OmpA, Mature OmpA, and the Signal Peptide of pro-OmpA-SecA was purified from SecA-overproducing cells as described (22). pro-OmpA was purified from pro-OmpA- overproducing cells as described (23). The preparation of OmpA from E. coli MH116OrecA (recA ompR101) was performed as reported (24). The signal peptide of pro-OmpA containing 21 amino acid residues was synthesized with a peptide synthesizer (Applied Biosystems Inc.). pro-OmpA, mature OmpA, and the signal peptide were dissolved in 50 mM Tris-C1 (pH 7.5) containing 1 mM dithiothreitol and 8 M urea.

SDS-PAGE and Immunoblot Analysis of SecY and SecE-A gel containing 12.5% acrylamide and 0.33% N,N’-methylenebisacryl- amide was used as described by Laemmli (25). All samples were applied to the gel without boiling. Immunoblot analysis was carried out as described (26).

Zn Vitro Transcription and Translation-Zn vitro transcription of the ompA gene was performed as described (27). The translation reaction was carried out in the presence of Tran3’S-label (0.46 mCi/ ml; 1 Ci = 37 GBq) as described (28). [35S]Met-labeled pro-OmpA was partially purified as reported (29).

In Vitro Protein Translocation-The translocation of [36S]pro- OmpA was performed as described (26) with slight modifications. The reaction mixture contained, in 25 pl, 5 pg of membrane vesicles, 0.75 pg of SecA, 2 mM ATP, 2 mM MgSO,, 5 mM NADH, 36S-pro- OmpA (2 X 10’ cpm), and 50 mM potassium phosphate (pH 7.5). In some experiments, 0.1-3 pg of non-radioactive pro-OmpA was added in addition to 36S-pro-OmpA. Energy donors, membrane vesicles, and pro-OmpA were separately preincubated at 37 “C for 2.5 min prior to the start of the assay. The assay was initiated by the addition of pro- OmpA to the mixture of membrane vesicles and energy donors. The translocated protein, which was resistant to proteinase K, was de- tected on an SDS-polyacrylamide gel by means of fluorography as described (30). Densitometric quantification of band materials was carried out with a Shimadzu CS-930 chromatoscanner. The total amounts of pro-OmpA and OmpA, which were translocated, were expressed as percentages of the input precursor protein. The numbers of methionine residues in pro-OmpA (6 residues) and OmpA (5 residues) were taken into consideration.

Assaying of ATPase Activity-ATPase activity was determined by means of a coupled spectrophotometric assay with pyruvate kinase and lactate dehydrogenase. The cuvette contained, in 2 ml, 50 mM potassium phosphate (pH 7.5), 1 mM dithiothreitol, 2 mM MgSOI, 3 mM phosphoenolpyruvate, 0.25 m M NADH, 10 mM KCN, 10 units of pyruvate kinase, 15 units of lactate dehydrogenase, and 50 pg of membrane vesicles. Potassium cyanide was added to inhibit NADH oxidase of membrane vesicles. To inhibit FoF1-ATPase, membrane vesicles were pretreated with DCCD, washed, and suspended in 50 mM potassium phosphate (pH 7.5) as described (20). The assay was started by the addition of 2 mM ATP, and oxidation of NADH was continuously monitored at 340 nm with a Shimadzu UV-3000 spec- trophotometer. SecA and pro-OmpA were subsequently added at 30 and 13 pg/ml, respectively. The amounts of ATP hydrolyzed were calculated by using a value of 6.22 for the millimolar absorption coefficient of NADH. Translocation ATPase represents the activity that increases upon the addition of pro-OmpA as described (8).

Determination of A* and ApH-Generation of A* (inside positive) and ApH (inside acidic) in membrane vesicles was monitored by following the fluorescence quenching of oxonol V and that of quina- crine, respectively, as reported (20). The reaction mixture contained, in 2 ml, 50 mM potassium phosphate (pH 7.5), 2 m M MgSO,, 1 p~

was started at 37 “c by the addition of 5 mM ATP or 1 mM NADH. oxonol V or quinacrine, and 100 pg of membrane vesicles. The assay

SecA, pro-OmpA, and CCCP were added as specified. M~terials-Tran~~S-label, a mixture of 70% [35S]methionine and

20% [35S]cysteine, 13.6 mCi/ml, was obtained from ICN. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), DCCD, phosphoenolpy- ruvate, NADH, and pyruvate kinase (500 units/mg of protein) were from Sigma. ATP was from Boehringer Mannheim. Lactate dehydro- genase (240 units/mg of protein) was from Biozyme. Proteinase K was from Merck. The anti-SecY and -SecE IgGs used were raised against synthetic peptides corresponding to the Metl-Arg’ region of SecY and the Lys’-Lyssl region of SecE, respectively, as described (31).

RESULTS

Effects of Overproduction of SecY and SecE on in Vitro Protein Translocation-Everted membrane vesicles were pre- pared from E. coli cells in which the overproduction of SecY and SecE was induced for various periods after the addition of IPTG. Quantitative immunoblot analysis of these mem- brane vesicles revealed that the expression of SecE and SecY became maximum at 90-120 min after the induction of over- production (Fig. 1, A and B ) . After 120-min induction, the amounts of SecY and SecE in membrane vesicles were about 20- and 200-fold, respectively, compared with those in mem- brane vesicles prepared from cells producing normal amounts of SecY and SecE, as reported (19). Hereafter, membrane vesicles prepared from SecY/SecE-overproducing cells with 120-min induction and those prepared from non-overproduc- ing cells are called “OP-membrane vesicles” and “normal membrane vesicles,” respectively.

Protein translocation was then determined with membrane vesicles as a function of the induction period in the presence of externally added SecA. The translocation of 35S-pro-OmpA did not increase with the increase in the amounts of SecY and SecE (Fig. IC). The possibility was then considered that the number of the possible secretory apparatus, including SecY and SecE, in membrane vesicles might have been in far excess of that of 35S-pro-OmpA, so that the overproduction was not properly reflected in the translocation activity. When excess amounts of non-radioactive pro-OmpA were added to the reaction mixture containing normal membrane vesicles, progressive inhibition of the translocation of 35S-pro-OmpA with the increase in non-radioactive pro-OmpA was observed. Essentially the same inhibition profile was observed when OP-membrane vesicles were used, excluding the possibility mentioned above (data not shown).

Effect of Overproduction of SecY and SecE on Translocation ATPase Activity-It has been reported that protein translo- cation accompanies the hydrolysis of ATP (8). This ATPase activity, defined as that of a translocation ATPase, requires SecA, a precursor protein, and membrane vesicles (8). It has also become clear that the hydrolysis of ATP is specifically coupled to the initial step of protein translocation rather than the entire process (23, 29, 32). The late step of OmpA trans- location is completed in the absence of ATP hydrolysis (23, 29, 32). The translocation ATPase activity in the presence of externally added SecA was determined with the various mem- brane vesicles used in Fig. lA. The activity increased with the increase in the induction period (Fig. IC). The highest activity was obtained when the overproduction was induced for more than 90 min. The translocation ATPase activity was then determined with both OP-membrane vesicles and normal membrane vesicles as a function of either the SecA concen-

Translocation ATPase and See Apparatus-dependent H" Flux 8195

A

+ SecY

+ SecE

-

Time (rnin)

1 50

-

0 1 . ' . , . . . . * . . '

Time (min) 50 100

0

FIG. 1. Effects of overproduction of SecY and SecE on the activity of translocation ATPase and pro-OmpA transloca- tion. Overproduction of SecY and SecE was induced by the addition of IPTG at 37 "C for the indicated periods. Membrane vesicles were then prepared. A, 20 pg of membrane was analyzed by SDS-PAGE, followed by immunoblotting with either anti-SecY or anti-SecE an- tibodies. B, the amounts of SecY (0) and SecE (0) detected on immunoblotting were densitometrically quantified and plotted as a function of the induction period. Values a t 120 min were taken as 100%. C, translocation ATPase (0) and translocation of [35S]pro- OmpA (0) were determined as described under "Experimental Pro- cedures" in membrane vesicles as a function of the induction period. The activity of translocation ATPase represents picomoles of ATP hydrolyzed per min per 10 pl of reaction mixture. The amounts of translocated proteins a t 37 "C for 5 min are expressed as percentages of the input pro-OmpA.

tration (Fig. 2 A ) or the pro-OmpA concentration (Fig. 223). The translocation ATPase exhibited similar profiles of de- pendence on both SecA and pro-OmpA with the two types of membrane vesicles. The activity obtained with OP-membrane vesicles, however, was significantly higher than that with normal membrane vesicles over the entire concentration ranges of SecA and pro-OmpA. These results, taken together, suggest that the overproduction of SecY and SecE specifically enhanced the activity in the earlier stage of protein translo- cation, which involves the SecA/pro-OmpA-dependent hy- drolysis of ATP, whereas the overproduction had little effect on the entire translocation activity.

Generation of the Proton Motive Force in OP-membrane Vesicles and Normal Membrane Vesicles-Generation of A* (inside positive) and ApH (inside acidic) in both types of membrane vesicles was examined by monitoring the fluores- cence quenching of oxonol V and that of quinacrine, respec-

tively, in the presence of ATP or NADH (Fig. 3). The level of A* generated upon the addition of NADH was slightly smaller in OP-membrane vesicles than in normal membrane vesicles (Fig. 3, E and F). This may suggest that OP-mem- brane vesicles are slightly more permeable to ions than normal membrane vesicles. On the other hand, the levels of A* and ApH generated by FoF1-ATPase upon the addition of ATP (Fig. 3, A-0) and that of ApH generated upon NADH addition (Fig. 3, G and H) were similar in both types of membrane vesicles. These results indicate that overproduction of SecY and SecE did not perturb the ability to generate the proton motive force. A* and ApH collapsed on the addition of a lipophilic anion, SCN-, and a membrane-permeable weak base, ammonia, respectively, as reported (33).

Addition of pro-OmpA in the Presence of SecA Causes the Collapse of ApH Generated in OP-membrane Vesicles- Whether or not protein translocation causes any changes in the permeability of membranes to protons was examined with OP-membrane vesicles. Changes in the magnitude of ApH may be compensated for if A* is present, since A* and ApH are interchangeable components of the proton motive force. For this reason, the effect of protein translocation on the proton permeability of membranes was examined by monitor- ing the generation of ApH in the absence of A*. The reaction mixture contained 50 mM KSCN to prevent the generation of A* (33) . The addition of ATP to the reaction mixture con- taining normal membrane vesicles (Fig. 4A) or OP-membrane vesicles (Fig. 4 , B-E) caused the quenching of quinacrine fluorescence, indicating that ApH was generated by FoFI- ATPase. The addition of SecA had no effect on the level of the quenching. The subsequent addition of pro-OmpA caused only a marginal change in the fluorescence intensity when normal membrane vesicles were used (Fig. 4.4). When OP- membrane vesicles were used, in contrast, the addition of pro- OmpA immediately caused a significant increase in the fluo- rescence intensity, indicating that ApH was partially col- lapsed (Fig. 4 B ) . Such a collapse was not observed in the absence of KSCN (data not shown), most probably due to the generation of A*, which may be converted to ApH. Since the pH of the reaction mixture remained constant during the assay, the collapse of ApH represents alkalinization of the intravesicular space. The fluorescence intensity increased to near the initial level on the addition of a proton conductor, CCCP. When pro-OmpA was added in the absence of exter- nally added SecA, only a slight change in ApH was observed (Fig. 4C). The extent of the ApH collapse was proportional to the amount of SecA added (Fig. 5A). The addition of mature OmpA instead of pro-OmpA did not lead to the collapse of ApH at all (Fig. 4 0 ) . The addition of urea, which was used to dissolve pro-OmpA or mature OmpA, had no effect on the generation of ApH either (data not shown). Furthermore, the addition of the pro-OmpA signal peptide in the presence of SecA had no effect on the fluorescence intensity (Fig. 4 E ) . The extent of the ApH collapse was dependent on the amount of pro-OmpA added (Fig. 5 B ) . Taken together, these results indicate that both SecA and pro-OmpA are involved in the ApH collapse. The effect of protein translocation on the level of A* was not examined, since pro-OmpA alone somehow caused quenching of oxonol V fluorescence.

The ApH Collapse Requires SecY-The ApH collapse was significantly suppressed when OP-membrane vesicles pre- treated with anti-SecY IgG were used, whereas pretreatment with non-immune IgG had no effect (Fig. 6), indicating that the collapse is dependent on SecY. Since generation of ApH was not observed from the beginning in the presence of anti- SecE IgG, the effect of this IgG on the pro-OmpA-induced

8196 Translocation ATPase and Sec Apparatus-dependent H+ Flux

FIG. 2. Effects of the concentra- tions of SecA and pro-OmpA on translocation ATPase activity. Translocation ATPase activity was de- termined with OP-membrane vesicles (0) or normal membrane vesicles (0) in the presence of 13 pg/ml pro-OmpA and various concentrations of SecA ( A ) or 30 pg/ml SecA, and various concentrations of pro-OmpA ( B ) . Translocation ATP- ase activity was determined as described under “Experimental Procedures” and expressed as picomoles of ATP hydro- lyzed per min per 10 pl of reaction mix- ture.

A B ATP uu ATP

DY/

C D ATP

(NH 4 )2 SO4 ATP 1 (NH4)2S04

DpH

S t k L

lmin

FIG. 4. The addition of pro-OmpA together with SecA caused the col- lapse of ApH generated on mem- brane vesicles containing overpro- duced SecY and SecE. The generation of ApH by FoF1-ATPase on either nor- mal membrane vesicles ( A ) or OP-mem- brane vesicles (B-E) was examined in the absence of A*. Where specified, SecA, pro-OmpA, mature OmpA, the sig- nal peptide of pro-OmpA, and CCCP were added at final concentrations of 60 pg/ml, 26 rg/ml (0.70 pM), 26 d m 1 (0.75 pM), 1.6 pg/ml (0.77 pM), and 10 p ~ , respectively. In C, 50 mM potassium phosphate (pH 7.5) was added in place of SecA.

1

A I 150

100

50

0

-I 1 I 1 B

E F NADH NADH

I KSCN I KSCN

ATP \ I - I r \

FIG. 3. Generation of A* (inside positive) and ApH (inside acidic) by membrane vesicles containing over- produced or normal amounts of SecY and SecE. Normal membrane vesicles ( A , C , E , and G ) or OP-mem- brane vesicles ( B , D, F, and H ) were prepared as described under “Experi- mental Procedures.” The generation of A* (A , B, E, and F ) and ApH (C, D, G, and H ) by FoF1-ATPase (A-D) or NADH oxidase (E-H) was determined by monitoring the fluorescence quench-

tively. Where specified, KSCN (final ing of oxonol V and quinacrine, respec-

concentration, 50 mM) or (NH4)2SO~ (fi- nal concentration, 10 DIM) was added to collapse A* and ApH, respectively.

:;u :/u sL 7

t t t t t t lmin

SeCA matureOmpA CCCP SecA signal peptide Cccp

collapse of ApH could not be studied. The effect of the when sufficient amounts of SecY and, probably, SecE, as well overproduction of SecY and SecE on the ApH collapse was as one of SecA, are present. examined in more detail with the various membrane vesicles ATP Is Essential for the Collapse of ApH-Since the above used in Fig. 1 as a function of the induction period. The mentioned results were obtained in the presence of ATP, collapse of ApH, which was dependent on externally added which was added to generate ApH through FoF1-ATPase, it SecA, increased with the increase in the induction period (Fig. was not clear whether or not ATP is essential for the collapse 7). These results indicate that the collapse becomes significant of ApH caused by pro-OmpA. In order to clarify this, the

Translocation ATPase and See

0 1 2 3 4 5 6 0 1 2 3 4 5 6 Time (min)

FIG. 5. Effects of the concentrations of SecA and pro-OmpA on the collapse of ApH. The collapse of ApH generated on OP- membrane vesicles was examined as described in the legend to Fig. 4 in the presence of either 26 pg/ml pro-OmpA and the indicated amounts of SecA ( A ) or 60 pg/ml SecA and the indicated amounts of pro-OmpA ( B ) . pro-OmpA was added 1 min after the addition of SecA. All recordings are overlaid.

A B

t t lmin SecA proOmpA

ATP /1 \At t t CCCP

FIG. 6. Collapse of ApH caused by pro-OmpA was SecY- dependent. OP-membrane vesicles were examined as to the collapse of ApH by pro-OmpA. SecA, pro-OmpA, and CCCP were added as described in the legend to Fig. 4. OP-membrane vesicles treated with 6.7 mg of nonimmune IgG ( A ) or 6.7 mg of anti-SecY IgG ( B ) at 0 "C for 30 min were used.

A Control B Omin C 30min

D 60min E 120min

FIG. 7. ApH collapse was observed only on membrane vesi- cles containing overproduced SecY and SecE. Membrane vesi- cles were prepared from cells in which the overproduction of SecY and SecE was induced for various periods. The ApH collapse in these membrane vesicles was examined as described in the legend to Fig. 4 as a function of the induction period (B-E), which is indicated in the figure. Assaying with normal membrane vesicles was also performed ( A ) . pro-OmpA was added at the time indicated by the arrow to each preparation of membrane vesicles in the presence (+) or absence (-) of SecA.

membrane vesicles were pretreated with DCCD to inhibit FoF1-ATPase. Generation of ApH in the absence of A\k was then performed by the addition of NADH. The DCCD-treated OP-membrane vesicles did not generate ApH upon the addi- tion of ATP but did generate it upon the addition of NADH (Fig. 8A) . When pro-OmpA was added after the addition of SecA, the collapse of ApH took place as observed in the case

Apparatus-dependent H' Flux

A " NADH

P

t ATP I

t t CCCP

Buffer ProOmPA

8197

SecA proOmpA

FIG. 8. Collapse of ApH caused by pro-OmpA was ATP- dependent. OP-membrane vesicles were incubated at 50 pg/ml in 2 ml of 50 mM potassium phosphate (pH 7.5) containing 2 mM MgSOa and 30 p~ DCCD on ice for 30 min. After a shift of the temperature up to 37 "C and the addition of KSCN and quinacrine at 50 mM and 1 p ~ , respectively, ApH was generated by the addition of NADH in the presence ( A and B ) or absence (C) of ATP. SecA, pro-OmpA, and CCCP were added as described in the legend to Fig. 4. In B, 50 mM potassium phosphate (pH 7.5) in place of SecA was added.

of the ATP-dependent generation of ApH. The collapse of ApH under these conditions was also SecA-dependent (Fig. 8B). When SecA and pro-OmpA were added in the absence of ATP, the ApH collapse was less than that in the presence of ATP (Fig. 8C), indicating that ATP is essential for the collapse of ApH.

DISCUSSION

Recent examinations of protein translocation into everted membrane vesicles of E. coli revealed that ATP hydrolysis is not coupled to the entire process of pro-OmpA translocation but rather specifically to the early step of the reaction (23, 29, 32). We showed in this paper that the SecA-catalyzed translocation ATPase activity exhibited in the presence of pro-OmpA and membrane vesicles increased with increases in the amounts of SecY and SecE in membrane vesicles (Fig. 1). In contrast, the entire translocation activity for pro-OmpA was not affected by the overproduction of SecY and SecE (Fig. 1). These results indicate that OP-membrane vesicles exhibit high activity in the earlier step of translocation. These results may also suggest that SecY, SecE, and SecA constitute only a part of the translocation apparatus in the membrane. While proteoliposomes reconstituted from purified SecY and SecE are active in protein translocation in the presence of SecA (lo), the activity was very weak compared with that of the intact membrane vesicles containing the same amounts of these proteins, suggesting the possible involvement of other membrane components in the translocation reaction. Genetic studies have demonstrated the involvement of SecD and SecF in protein secretion in E. coli (3). Results indicating the involvement of SecD in the late step of protein translocation were recently obtained (34). The possible participation of a membrane protein, band 1, in protein translocation is also

8198 Translocation ATPase and See Apparatus-dependent Hi Flux

suggested (35). Reconstitution studies directly demonstrating the functions of these proteins have not been reported yet, however.

The addition of pro-OmpA to OP-membrane vesicles in the presence of SecA and ATP caused the partial collapse of ApH (Fig. 4). The collapse of ApH depended on pro-OmpA, SecA (Fig. 5 ) , SecY (Fig. 6), and ATP (Fig. 8). SecE dependence could not be demonstrated because of the inhibition of ApH generation by anti-SecE antibody. The ApH collapse took place in OP-membrane vesicles but not in normal membrane vesicles, suggesting that the early event of protein transloca- tion causes the ApH collapse. The very rapid collapse of ApH after the addition of pro-OmpA suggests that neither gradual accumulation of a translocation intermediate nor that of an incorrectly translocated one induces the ApH collapse in OP- membrane vesicles. Since both OP-membrane vesicles and normal membrane vesicles exhibited similar activity of pro- OmpA translocation, it seems unlikely that the ApH collapse in OP-membrane vesicles is caused by protonation of OmpA after its translocation. Taken together, these results indicate that the collapse of ApH most likely represents the outflow of protons from membrane vesicles, which is associated with the early stage of protein translocation. It should be men- tioned that the late stage of pro-OmpA translocation also accompanies the proton transfer (36).

Simon and Blobel (15) reported that the release of a nascent peptide from ribosomes attached to the ER membrane causes a significant increase in the ion conductance of the membrane. It is assumed that a protein-conducting channel opens upon the release of nascent peptides and conducts various ions as well. A similar channel was suggested to be present in E. coli recently (16). It is not known, however, whether or not any Sec proteins are involved in the channel activity. The opening of such a channel should immediately result in the complete collapse of the proton motive force. It should be noted that opening of the channel was reported to be induced on the addition of the signal peptide in the absence of externally added SecA (16), whereas the ApH collapse shown in the present work required the external addition of SecA and was not induced by the signal peptide. It seems unlikely, therefore, that the collapse of ApH observed in the present work is related to the reported channel activity.

The mechanism whereby the early step of protein translo- cation causes the ApH collapse is not certain at present. The outflow of protons may be caused by possible protein/proton antiporter activity of the translocation apparatus as discussed (14). However, the lack of parallelism between the ApH collapse and pro-OmpA translocation may suggest that this is less likely. The ApH collapse can be caused by either a specific increase in proton permeability of membranes or enhancement of membrane leakiness to ions. Whichever the case is, the results shown in this paper indicate that the ApH collapse as well as the hydrolysis of ATP represents an important property associated with the activation of Sec apparatus at the early stage of protein translocation. It seems likely that the ApH collapse is caused by the conformational

change of Sec apparatus at the early stage of protein trans- location but not the complete translocation of pro-OmpA. SecA, which interacts with both membranes and SecY/SecE, was shown to change its conformation upon the interaction with pro-OmpA or ATP (37).

Protein translocation was recently reported to make the membrane of E. coli permeable to halide anions (17). The relation of the ApH collapse to this reported results is not clear, since halide anions were not included in our assay mixtures.

Acknowledgment-We thank Iyoko Sugihara for the excellent sec- retarial support.

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