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

Vol. 268, No. 13, Isaue of May 5, pp. 9442-9447,1993 Printed in U.S.A.

Charged Residues Render Pro-OmpA Potential Dependent for Initiation of Membrane Translocation*

(Received for publication, November 12, 1992, and in revised form, January 4, 1993)

Bruce GellerSB, Heng-Yi ZhuT, Shiyuan Chengn, Andreas Kuhnll**, and Ross E. DalbeyTSS From the llDepartment of Chemistry, the Ohio State University, Columbus, Ohio 43210, the I( Department of Applied Microbiology, University of Karlsruhe, 0-75 Karlsruhe, Germany, and the $Department of Microbiology and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, Oregon 97331-3804

We have examined the effects of positively and neg- atively charged residues on the translocation of outer membrane protein A precursor (pro-OmpA) across the bacterial inner membrane. Pro-OmpA does not trans- locate across the membrane when 2 positively charged residues are inserted immediately after the leader pep- tide, whereas it does insert when 2 neutral or nega- tively charged residues are introduced. Using a cell- free translocation system, we show that the membrane potential stimulated the rate of initial insertion of pro- OmpA with negatively charged residues, inhibited pro- OmpA with positively charged residues, and had no effect on neutral pro-OmpA. Thus, acidic residues ren- der pro-OmpA potential-dependent for loop formation, which then initiates the translocation process.

Proteins exported to the periplasm or to the outer mem- brane of Escherichia coli almost always contain cleavable leader (or signal) sequences. In contrast, the majority of inner membrane proteins are synthesized with uncleavable signal peptides (Wickner and Lodish, 1985). Genetic studies have established that both types of signal sequences are necessary for translocating proteins into or across the plasma membrane (Emr and Silhavy, 1983; Michaelis and Beckwith, 1982; Ban- kaitis et al., 1985; Benson et al., 1985; Carlson and Botstein, 1983; Dalbey and Wickner, 1987). However, in most cases tested so far, a bacterial signal sequence is not sufficient to export an attached cytosolic protein. This was first demon- strated by Beckwith and colleagues, who showed that the lam B leader sequence could not export /%galactosidase across the inner membrane (Silhavy et al., 1977; Bassford et al., 1979).

There is now compelling evidence that many cytosolic proteins contain sequences that prevent transport across the bacterial membrane. The best example of such a cytoplasmic protein was investigated by Summers and Knowles (1989). In

* This work was supported by National Science Foundation Grant DCB-9020759, an American Cancer Society Junior Faculty award, a seed grant from the Office of Research and Graduate Studies, a Basil O’Conner starter grant from the March of Dimes, a pilot research grant from the American Cancer Society (Ohio State University) and a grant from the American Cancer Society (Ohio Division). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by grants from the Medical Research Foundation of Oregon, National Science Foundation Grant DMB 86-18186, and the American Cancer Society Grant JFRA-200.

** Supported by Grant 3.533-0.86 from the Swiss Science Foun- dation and from the Deutsche Forschungsgemeinschaft.

$$ To whom correspondence should be addressed. Tel.: 614-292- 7451.

this study, the p-lactamase leader peptide was insufficient to secrete chicken muscle triosephosphate isomerase, which is normally cytoplasmic. However, when the arginine at position 3 in the triosephosphate isomerase was replaced by a Iysine or a neutral residue, the cytosolic protein was secreted. Fur- thermore, Li et al. (1988), Yamane and Mizushima (1988), and MacIntyre et al. (1990) have shown that positive charges block export when introduced directly after the leader se- quences of exported proteins. Similar effects have also been found with leader peptidase, a transmembrane protein of the plasma membrane of E. coli, which lacks a cleavable signal sequence. We have identified a polar segment, termed a “translocation poison” domain, that prevents the preceding apolar domain from functioning as a signal peptide (von Heijne et al., 1988). Recently, we have shown that the posi- tively charged residues within this polar region have an inhib- itory effect on translocation (Laws and Dalbey, 1989). It is not clear why these positive charges block export. We also do not know how close the charges have to be to a signal peptide to exert a negative effect on translocation. The purpose of this study is to address these issues and also to examine the effects of negatively charged residues on export.

We chose as a model system the well characterized outer membrane protein A (OmpA).’ This protein is synthesized as a precursor, called pro-OmpA, with a leader sequence of 21 residues. Its translocation across the inner membrane requires SecA and SecY both in uiuo (Wolfe et al., 1985) and in vitro (Cabelli al., 1988; Cunningham et al., 1989). Site-directed mutagenesis was used to introduce either 2 positively charged residues, 2 neutral residues, or 2 negatively charged residues. Translocation was completely blocked when 2 arginine resi- dues were inserted at positions 1 and 2 in the mature region and was not inhibited when either 2 glycine or 2 glutamic acid residues were inserted at the same position. We present evidence using an in vitro translocation system that the rate of processing of pro-0mpA is dependent on the membrane- potential when charged residues are introduced after the leader peptide. These findings suggest that the membrane electrical potential affects mutant OmpA processing and translocation via direct interaction of the charged amino acids of the precursor with the potential gradient, or, alternatively, via effects on other component(s) of the secretion and proc- essing machinery.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids-E. coli HJM114 (Alac- pro)F’(lacpro), JM103(Alacpro) thi, strA, supE, end& sbcB, hsdR, traD36, proAB, laciqZM15), and Dk8 (bglR, thi-1, rel-1, HfrPO1,

The abbreviations used are: OmpA, outer membrane protein A; PAGE, polyacrylamide gel electrophoresis; CCCP, carbonyl cyanide p-chlorophenylhydrazone.

9442

T h e Role of Charged Residues on Protein Translocation 9443

AuncB-uncC) i1v::TnlO) were from our collection. JF699 (proC24, ompA252, his-53, purE41, ilv-277, met-65, lacY29, xyl-14, rpsL97, cycAl,cycB2, tsx-63, X-) was obtained from Dr. William Wickner. The pQN plasmids containing the OmpA gene were described in Kuhn et al. (1987). The pT713 plasmid with the T7 promoter was purchased from Bethesda Research Laboratories (BRL). The OmpA gene was cloned into the pT713 plasmid in the following fashion. The OmpA gene was excised from the M13 mp19 containing the pro- OmpA coding region (Kuhn et al., 1987) by digestion with HindIII and EcoRV. After the OmpA gene fragment was purified by electro- phoresis in 0.7% agarose, i t was inserted into the pT713 vector previously cut with SmaI and HindIII.

Materials-Trypsin (~-1-tosylamido-2-phenylethy1chloromethy1 ketone treated) and soybean trypsin inhibitor were from Worthing- ton. Proteinase K was from Boehringer Mannheim. Phenylmethyl- sulfonyl fluoride was from Sigma. "Translabel," a mixture of 85% [35S]methionine and 15% [35S]cysteine (1000 Ci/mmol), was from ICN. [35S]dATP and DNA polymerase I (Klenow fragment) were from New England Nuclear and Boehringer Mannheim, respectively. Oligonucleotides were synthesized at the Biochemical Instrument Center a t Ohio State University.

Site-directed Mutagenesis-Oligonucleotide-directed mutagenesis was performed as described in Dalbey and Wickner (1987). Restric- tion enzymes were purchased from BRL, New England Biolabs, and Boehringer Mannheim. All enzyme reactions and DNA manipula- tions were performed as described by Maniatis et al. (1982). The method of Cohen et. al. (1973) was used for DNA transformation.

Media, Labeling, and Protease Accessibility Experiments-M9 min- imal media (unless indicated) was prepared as described in Miller (1972) and was supplemented with 0.5% fructose and 50 pg/ml of each amino acid except methionine and cysteine. For radioactive labeling studies, the cells bearing a plasmid encoding pro-0mpA were grown in M9 media to the early log phase at 37 "C. At A b m = 0.2, the plasmid-encoded proteins were expressed by adding 0.2% arabi- nose. The cells were then labeled with [35S]Translabel for 30 s and incubated with non-radioactive amino acids for the indicated times. For protease accessibility studies, cells were labeled for 30 s and subsequently incubated with 500 pg/ml of non-radioactive methio- nine. At the indicated times, an aliquot (0.5 ml) of cells was trans- ferred to an equal volume of 40% sucrose, 60 mM Tris, and 20 m M EDTA, pH 8.0 (ice-cold), to permeabilize the outer membrane of the cells. Samples were then analyzed for proteolysis and subjected to immunoprecipitation (Wolfe et al., 1982), SDS-PAGE, and fluorog- raphy (Ito et al., 1980).

In Vitro Protein Trans&~cation-[~~S]Pro-OmpA was synthesized in a cell-free reaction (Gold and Schweiger, 1971) programed with the pT7 plasmids containing the OmpA gene, and supplemented with 333 units/ml T7 RNA polymerase (BRL). After 1 h a t 37 "C, the reaction was stopped with 250 pg/ml chloramphenicol, cooled to 4 "C, and desalted twice, just prior to use in the translocation reaction, on G-25 Sephadex (Pharmacia LKB Biotechnology Inc.) columns equil- ibrated with 50 mM Tris-HCI, pH 7.5, 1 mM dithiothreitol, 1 mg/ml bovine serum albumin. Inverted inner membrane vesicles were pre- pared as described (Geller and Green, 1989). The translocation reac- tions contained 0.4 mg/ml membrane vesicle proteins, 2 mM spermi- dine, 8 mM putrescine, 40 mM KC1, 5 mM Mg(acetate)2, 0.67 mg/ml bovine serum albumin, 50 mM Tris-HC1, pH 7.5, and 1 mM dithio- threitol. Where indicated, ATP and NADH were included a t 10 mM each, and nigericin and carbonyl cyanide rn-chlorophenylhydrazone (CCCP) at 1 and 5 pM, respectively. A control reaction without ATP contained 4% glycerol and 0.4 mg/ml glycerol kinase. At 0, 1, 2, 5, and 15 min of incubation a t 40 "C, aliquots were removed from the reactions and treated with or without 0.4 mg/ml proteinase K a t 0 "C for 15 min. After inhibition of the protease with phenylmethylsulfonyl fluoride, the amounts of pro-OmpA and OmpA were analyzed by SDS-PAGE and fluorography, and quantified by laser densitometry as described (Geller and Green, 1989). The amount of processing that occurred in the reactions without ATP was subtracted from the amount processed in the reaction that included energy. The energy- independent processing of wild-type and each mutant pro-OmpA increased from 0% (at zero time) to a maximum of 3% (at 15 min) of the total pro-OmpA added to the reaction. Typically, the amount of energy-independent processing was less than 1% of the total pro- OmpA added at 0-5 min time points, and about 2% a t 15 min for all types of pro-0mpA. The cause of this small amount of energy- independent processing was not investigated, but may be due to right- side-out vesicles, or a residual amount of ATP from the protein synthesis reaction.

RESULTS

To determine the effects of positively and negatively charged residues on the membrane translocation of a secretory protein, we chose pro-OmpA as a model system. Pro-OmpA is synthesized with a typical leader peptide that contains 2 basic amino acids near its amino terminus, followed by a long stretch of apolar amino acid residues that ends at the cleavage site. The first 20 amino acid residues in the mature domain have lysine residues a t positions +3 and +12 (relative to the leader peptidase cleavage site), and aspartic acid residues at positions +4 and +20. Oligonucleotide-directed mutagenesis was used to introduce either 2 positively charged residues, 2 neutral residues, or 2 negatively charged residues immediately after the leader sequence a t positions +1 and +2 of the mature protein. In this study, we measured the kinetics of processing in the E. coli strain JF699 lacking the chromosomal copy of OmpA. Fig. 1A shows that pro-OmpA RR was not processed even at a 10-min chase time. In contrast, neutral or negatively charged residues do not interfere with translocation when they are located at the same positions as the arginine residues. Pro-OmpA GG (Fig. 1B) and pro-0mpA EE (Fig. 1C) are rapidly converted to OmpA in a 5-s chase, as is seen with

,leader peptide-., ,"malure region, processing

1. Pro-OmpA RR

2. Pro-OmpA GG

3. Pro.OmpA EE

4. Pro-OmpA RG

5. Pro-OmpA GR

6. Pro-OmpA R5

MKKTAIAIAVALAGFATVAOA RRKDNTWYTGAK .... +325 - MKKTAIAIAVALAGFATVAOA GKDNTWYTGAK .... + 325 +

MKKTAIAIAVALAGFATVAOA EEKDNTWYTGAK. ... + 325 +

MKKTAIAIAVALAGFATVAOA EKDNTWYTGAK .... 1325 - MKKTAIAIAVALAGFATVAOA GBKDNTWYTGAK. ,. + 325 - , +

MKKTAIAIAVALAGFATVAOA APKORTWYTGAK ..... +325 -

.21 + 1 + 5 +10

A. Pro-OmpA RR

5" 1' 5' 10'

D. Pro-OmpA RG -

0- P-

B. Pro-OmpA GG C. Pro-OmpA EE ..-3F"- -

0- P-

0- P.

5" 1' 5' 10' 5" 1' 5' 10'

E. Pro-OmpA GR F. Pro-OmpA R5 .">- .,. - .

P 0

5" 1' 5' 10' 5" 1' 5 ' 10' 5" 1' 5' 10' FIG. 1. Processing of pro-OmpA mutants with charged res-

idues introduced after the leader sequence. JF699 (OmpA- strain) bearing the pQN plasmid encoding pro-OmpA RR ( A ) , GG ( B ) , EE (C), RG (D), GR ( E ) , or R5 (F) was grown at 37 "C to the mid-log phase in M9 medium containing 0.5% fructose and adenine (1 mg/ml), and the 19 amino acids except cysteine. After induction of pro-OmpA with 0.2% arabinose for 30 min, the proteins were labeled by incubating cells (2 ml) with 10 pl of [35S]Translabel for 30 s and then treating with non-radioactive cysteine (500 pg/ml final concentration). At the indicated times, cells (0.5 ml) were removed and transferred to an equal volume of ice-cold 20% trichloroacetic acid. The samples were then analyzed by OmpA immunoprecipitation, SDS-PAGE and fluorography, as described under Experimental Pro- cedures." p, in the figure, indicates pro-OmpA; o depicts mature OmpA. The first 12 residues in the mature domain of the wild-type pro-OmpA are APKDNTWYTGAK.

9444 The Role of Charged Residues on Protein Translocation

wild-type (data not shown). We also examined in a systematic fashion the effects of the position of a positively charged residue on the translocation of pro-0mpA. As can be seen in Fig. 1, D and E, respectively, pro-OmpA RG was not processed during a 10-min chase, and the processing of pro-OmpA GR was drastically inhibited. There was no measurable effect on translocation when arginine was introduced at position +5 (Fig. IF) or at position +8, or at +11 (data not shown).

Protease accessibility technique (Dalbey and Wickner, 1986) was used to directly establish protein translocation across the membrane. For these studies, we switched to the strain HJM114 since its outer membrane is easy to permea- bilize by osmotic shock treatment. This strain also has the advantage that we can use its chromosomal-encoded OmpA as a positive control for permeabilization. HJM114 synthesiz- ing the pro-OmpA mutant was pulse-labeled for 30 s and chased for the indicated times. At each chase point, an aliquot was removed, treated with 40% sucrose, 60 mM Tris and 20 mM EDTA, pH 8.0, to permeabilize the outer membrane, and then digested with trypsin (500 pg/ml) at 0 "C. Samples were then immunoprecipitated with OmpA antiserum and analyzed by SDS-PAGE and fluorography. In cells synthesizing pro- OmpA RR, we observed two immunoprecipitable species at a 5-s chase point (Fig. 2). The mature OmpA obviously corre- sponds to the chromosomally encoded wild-type copy and is accessible to the protease. The mutant pro-OmpA RR with its higher molecular weight was not digested by the protease even after 10 min of chase time. Only when the membrane bilayer was disrupted by the addition of detergent was the mutant protein digested (see 5" and 10' lysis lanes).

Since the membrane potential is more positive on the periplasmic side of the membrane, positively charged residues in precursor proteins may interfere with the translocation process because they cannot overcome the membrane poten- tial. To test this idea, we examined pro-0mpA RR, GG, and EE using an in vitro translocation system where the energy requirements could be manipulated. Recently, it has been shown that there are two consecutive energetically distinct steps (Geller and Green, 1989; Tani et al., 1989; Schiebel et al., 1991) in the translocation of wild-type pro-OmpA. ATP initiates the first step to insert the amino terminus of OmpA exposing the leader peptidase cleavage site to the periplasm. The second step, which completes the transfer of the protein across the membrane, is triggered by either ATP or the

Pro- RR

pro-OmpA- "

Chase Time 5" 1' 5 ' 10' 5"lO' Protease - + - + - + - + + +

+lysis FIG. 2. Protease-accessibility of a positively charged pro-

OmpA mutant. E. coli strain HJM114 (OmpA+ strain) expressing pro-OmpA RR was grown to the mid-log phase and incubated with arabinose (0.2% final concentration) for 30 min. Proteins were pulse- labeled with [35S]methionine for 30 s and then chased with non- radioactive methionine (500 pg/ml) for the indicated times. At each chase point, an aliquot (0.5 ml) was transferred to an equal volume of ice-cold buffer A (40% sucrose, 20 mM EDTA, and 60 mM, pH 8.0) to permeabilize the outer membrane. Samples were incubated at 0 "C with or without trypsin (500 pg/ml) for 60 min. Another portion of cells was incubated with trypsin (500 pg/ml) and 2% Triton X-100 for 60 min. After the addition of soybean trypsin inhibitor (1.25 mg/ ml) and phenylmethylsulfonyl fluoride (5 mM), samples were analyzed by immunoprecipitation, SDS-PAGE, and fluorography.

transmembrane electrochemical potential. Pro-OmpA was synthesized in a cell-free reaction and mixed with inverted plasma membrane vesicles that were prepared from an unc- mutant. These vesicles lack the FIFo-ATPase and can only generate a membrane potential with substrates (such as NADH) that act by way of the electron transport chain and not with ATP. The initial insertion and cleavage of the leader peptide of wild-type pro-OmpA was nearly unaffected by the electrochemical potential (Fig. 3A) , as reported previously (Geller and Green, 1989). Mutant pro-OmpA RR, with argi- nine at +1 and +2, was very slowly inserted and processed in an ATP-dependent manner, in the absence of the electro- chemical potential (Fig. 3B, 0). However, in the presence of the electrochemical potential (Fig. 3B, O), generated by the addition of NADH via the respiratory electron transport chain, insertion and processing of the RR mutant was impeded. This suggests that the potential has an inhibitory effect on loop formation. Fig. 4B shows that the RR mutant was digested by protease added from the outside of the mem- brane vesicles, demonstrating that this mutant does not com- pletely translocate across the membrane. It is intriguing that the effects of the pmf on processing and proteinase K protec- tion are different for the RR mutant. Apparently, this mutant is only capable of an inefficient insertion of the amino- terminal transmembrane loop.

-=c Q

E 0 n W m v, W 0 0 [li L1

IL+" 0.00

FIG. 3. Kinetics of in vitro processing of wild-type and pro- OmpA mutants with either 2 positively charged residues, 2 negatively charged residues, or 2 neutrally charged residues introduced after the leader sequence. At the indicated times, the amount of OmpA that was produced by the processing of pro-OmpA was determined (see Experimental Procedures"). The values were calculated by dividing the amount of OmpA by the total pro-OmpA + OmpA. Reactions contained either ATP (0) alone, or ATP + NADH (0). A , wild-type pro-OmpA. B, pro-OmpA RR. C, pro-OmpA EE. D, pro-OmpA GG.

T h e Role of Charged Residues on Protein Translocation 9445

-IE 0.20 0.00 0.10 0.00

0.20

0.10

0.00

m 0.50

nyE (mk)

FIG. 4. Kinetics of in vitro translocation of wild-type and mutant pro-OmpA. The same samples used for the data in Fig. 3 were analyzed with proteinase K. The OmpA protected by the mem- brane vesicles was quantified from reactions with ATP (0) and with ATP + NADH (0). The values are expressed as the ratios of protease- protected OmpA/(total pro-OmpA + OmpA) in the non-proteolyzed sample shown in Fig. 3. A, wild-type pro-OmpA. B, mutant RR. C, mutant EE. D, mutant GG.

In contrast to the pro-OmpA RR, the rate of insertion and processing of pro-OmpA EE was stimulated by the eletro- chemical potential (Fig. 3C). The intriguing result is that the electrochemical potential has the opposite effect on the EE mutant than it does on RR mutant. Moreover, there is a more pronounced membrane potential effect on the completely translocated pro-OmpA EE protein (Fig. 4C), compared to the wild-type protein (Fig. 4A). These data are consistent with an electrophoretic effect in which the potential helps transfer the amino terminus of mature OmpA across the membrane when negatively charged residues are located after the leader peptide. A second possible interpretation of these results with 2 glutamic acid residues is that the effects may not result from the introduction of negative charges but from the removal of proline at position 2, which is known to render pro-OmpA potential-dependent (Lu et al., 1991). Therefore, as a control, we examined the effects of the electrochemical potential on the GG mutant. Insertion and processing of pro- OmpA GG was not significantly stimulated by the electro- chemical potential (Fig. 3 0 ) .

To test whether it is the electrical potential that stimulates translocation of pro-OmpA EE, and not the PH gradient, we used the drugs CCCP and nigericin. CCCP collapses both the pH and electrical gradients; nigericin collapses only the pH gradient. The addition of the protonophore CCCP (Fig. 5A, M), only slightly (less than 2-fold) decreased the rate of

Qa E n 0

W v, v, W 0 0 CY a

0.60-

0.50--

A WILD TYPE

0.ooY ! I

0.60 I R EE

0.50.-

0.40

0.20

0.10::

o.oo&'=P- : 0 2 4 6 8 10 12 14 16

TIME (min)

FIG. 5. Kinetics of in vitro processing. A, wild-type pro-OmpA. B, pro-0mpA EE. At the indicated times, the amounts of OmpA that were produced by the processing of pro-0mpA were determined as described in Fig. 3. The reactions contained ATP (0), ATP + NADH (O), ATP + NADH + CCCP (m), and ATP + NADH + nigericin (A).

processing of wild-type pro-OmpA over the first 2 min as compared to the rate with the electrochemical potential (Fig. 5A, 0). In contrast, the rate of processing of pro-OmpA EE in the presence of CCCP was greatly (more than 10-fold) reduced (Fig. 5B, M) compared to the rate with the electro- chemical potential. This reduced rate with CCCP was the same as the rate without added NADH (Fig. 5B, 0). On the other hand, the addition of nigericin, which collapses the ApH by a transmembrane, stoichiometric exchange of K+ for H+, had no effect on either wild-type or pro-OmpA EE processing (Fig. 5, A and B, A).

The effects of CCCP and nigericin on the completion of translocation of the pro-OmpA chain are shown in Fig. 6, A and B. The translocation rate over the first 2 min was reduced about 6-fold for the wild-type protein with CCCP (D) or was reduced 6-fold when NADH was not added (0), as compared to the rate with the electrochemical potential (0). CCCP or the absence of NADH caused an even more significant de- crease (greater than lo-fold) in the rate of complete translo- cation of the EE protein (Fig. 6B). In contrast, nigericin had very little effect on the rate of complete translocation of either protein (Fig. 6, A and B, A), decreasing the initial rates of wild-type and EE proteins by 30 and 50%, respectively, over the first 2 min, but not at all after 5 min.

AS a control to confirm that the nigericin eliminated the pH gradient, the ApH was measured directly using a pH responsive, fluorescent probe. In this study, the same concen- tration of membranes, nigericin, and NADH was used as in the experiments shown in Figs. 5 and 6. Table I show that NADH created a transmembrane ApH that was eliminated by either CCCP (experiment 2) or nigericin (experiment 3). Because CCCP did not decrease further the ApH after niger- icin addition (experiments 3 and 4), this shows that nigericin was as effective in eliminating the ApH as CCCP. Further- more, the data reveal that when nigericin was added before NADH (experiment 4), no pH gradient was formed. These results indicate that nigericin completely eliminated the ApH in these studies.

9446 T h e Role of Charged Residues on Protein Translocation

0.60+ A WILD lYPE

/

Q o.oocL, L W

0.00 0 2 4 6 8 1 0 1 2 1 4

TIME (min)

FIG. 6. Kinetics of in vitro translocation. A, wild-type pro- OmpA. B . pro-0mpA EE. The samples used for the data in Fig. 5 were analyzed with proteinase K for protein translocation. The amount of protease-resistant OmpA was quantified as described in Fig. 4. The symbols are the same as in Fig. 5.

DISCUSSION

We have analyzed the effects of charged residues on protein translocation when introduced after the leader peptide of pro- OmpA. Translocation is blocked when positively charged res- idues are introduced (Fig. U), while neutral (Fig. 1B) or negatively charged (Fig. IC) residues do not have a measura- ble effect on insertion. In addition, other recent studies where a positively charged cluster, comprised of Lys-Arg-Arg-Glu- Arg, is introduced into the early mature region of pro-OmpA,Z show that a region of about 20 residues is important for translocation. Positive charges cause interference in export presumably because they prevent the first 20 residues in the mature region of pro-OmpA from forming the critical loop structure that is essential for initiating translocation of the mature region of the protein (Summers et al., 1989). There is strong evidence that loop formation occurs during the early translocation steps of pro-OmpA (Geller and Green, 1989; Tani et al., 1989; Driessen, 1992). The transmembrane loop is comprised of the leader peptide and approximately 20

R. Dalbey and A. Kuhn, unpublished data.

residues of the mature region (Schiebel et al,, 1991). A similar region, termed “export initiation domain,” has been proposed by von Heijne from studies in which 6 consecutive lysines blocked translocation of the carboxyl-terminal domain of leader peptidase (Andersson and von Heijne, 1991).

Protein export of wild-type pro-OmpA across the mem- brane requires both ATP hydrolysis (Chen and Tai, 1985; Geller et al., 1986) and the membrane electrochemical poten- tial (Zimmermann and Wickner, 1983); precursors to OmpA do not cross the membrane either in the absence of ATP or in presence of uncouplers that dissipate the membrane elec- trochemical potential. I n vitro studies (Geller and Green, 1989) have shown that the initial insertion of the amino- terminal domain of wild-type pro-OmpA, which exposes the leader peptidase cleavage site, is driven by ATP hydrolysis and is unaffected by the membrane potential. The role of the potential is to stimulate the complete transfer of the protein across the membrane. These data show that there is no electrophoretic effect for the wild-type protein (which has no net charge in the first 20 mature residues).

In contrast, the membrane electrochemical potential can contribute to the rate of membrane insertion of the pro-OmpA mutants when charged residues are introduced. We found that the slow rate of in vitro processing of pro-OmpA RR with 2 arginines at +1 and +2 (compared to the wild-type) was impeded by an electrochemical potential (Fig. 3). Conversely, the rate of insertion and processing of pro-OmpA with 2 negatively charged residues after the leader peptide was stim- ulated by the electrochemical potential. Acidic residues, there- fore, render pro-OmpA membrane potential dependent for the formation of the translocation intermediate that exposes the leader peptidase cleavage site to the periplasm. Moreover, there was a large potential-dependent stimulation in the amount of the fully translocated negatively charged mutant pro-OmpA, compared to the wild-type. These findings suggest that an electrophoretic mechanism contributes to the force which drives the charged pro-OmpA mutants across the mem- brane. This is also supported by the experiments with niger- icin and CCCP shown in Figs. 5 and 6. Because the rate of processing of the negatively charged pro-OmpA EE was sig- nificantly reduced in the absence of both A$ and ApH, whereas it was greatly stimulated with either the complete electrochemical potential or A$ alone, it is inferred that the stimulation is the result of the A$. To our knowledge, this is the first evidence of an electrophoretic mechanism of protein translocation.

We believe the membrane electrical potential affects mu- tant OmpA translocation and processing via direct interaction of the charged amino acids of the precursor with the potential gradient. Another possibility is that the effect is on other

TABLE I NADH-dependent membrane enegization

The membrane ApH was measured by fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) as described (Klionsky et al., 1983), except the buffer was 50 mM potassium morpholinopropane sulfonate, 15 mM MgC12, 40 mM KCl, pH 7.3, 1 mg/ml bovine serum albumin, 1 mM dithiothreitol, 1 mM spermidine, and 8 mM putrescine. All measurements were made with an Aminco-Bowman spectropho- tofluorometer. The initial fluorescence of the membranes (0.4 mg protein/ml) in buffer was set to 0%. ACMA (2 PM) was added, and the level of the fluorescence set to 100%. The following were added in sequence: either 10 mM NADH, 1 p~ nigericin, and 5 p~ CCCP (experiment 31, or 1 p M nigericin, 10 mM NADH, and 5 p~ CCCP (experiment 4). A control experiment without nigericin (experiment 2) was done by sequentially adding NADH and CCCP.

ApH as a function of relative fluorescence intensity Experiment Membranes

ACMA NADH Nigericin CCCP Nigericin NADH CCCP

1 - 100 101 106 106 2 + 100 I Not added 119 3 + 100 8 121 122 4 + 100 120 119 118

The Role of Charged Residues on Protein Translocation 9441

component(s) of the secretion and processing machinery, e.g. LepB, SecA, SecE/SecY. Alternatively, ion gradients may influence the interaction of the precursor with secretion ma- chinery components directly. A direct effect of the potential on leader peptidase activity seems unlikely because positively and negatively charged mutants behave oppositely with re- spect to processing as a function of the gradient.

These data suggest that the inhibition of insertion of the RR mutant in the presence of a membrane potential is due to the inhibition of signal peptide loop penetration through the membrane. However, we cannot distinguish between this pos- sibility and another where the loop inserts transiently, but does not remain stably inserted due to the electrical potential. In either case, the kinetics of interaction and processing of the loop by leader peptidase, and the rate of translocation of the OmpA chain, would be slowed. One could also imagine that the RR mutation could cause a change in the secondary and tertiary structure of pro-OmpA and impair the ability of the precursor to interact with the export machinery or leader peptidase. We feel it is unlikely that the RR mutation could impair leader peptidase processing of the precursor since argininyl amino acids are found at these positions in pre- proteins (von Heijne, 1983).

While the generality of the electrophoretic effect to other proteins is unknown, P-lactamase (Bakker and Randall, 1984) and M13 procoat protein (Kuhn et al., 1990) apparently are not translocated primarily by electrophoresis. Furthermore, a model secretory protein without charged residues in the ma- ture domain was translocated in an electrochemical potential- dependent manner (Kato et al., 1992), suggesting that the potential affects the translocation apparatus. If electropho- resis plays a role in protein translocation, it may not neces- sarily be a dominant force. However, we believe that electro- phoresis may be a common driving force for translocation because exported proteins in bacteria often have a net nega- tive charge in the carboxyl-terminal region of the leader peptide and in the first few positions of the mature sequence (von Heijne, 1986).

The stimulating effect of the membrane potential on pro- OmpA with negatively charged, but not positively charged, residues helps explain why acidic residues can cross the mem- brane easier than basic amino acids. Thermodynamics pre- dicts that the energy cost for burying two negative charges within the lipid bilayer would be greater than that for 1 arginine residue (Engelman and Steitz, 1981). However, we found that these negative charges did not inhibit protein translocation of pro-OmpA (Fig. IC). This shows, at least with a small number of negative charges, that the stimulating effect of the membrane potential can outweigh the inhibitory effect of burying the charges within the bilayer. Clearly, however, the membrane potential is not the sole factor be- cause the addition of many negative charges after the signal peptide can reduce or block the rate of translocation of proteins across the membrane (Laws and Dalbey, 1989; Nils- son and von Heijne, 1990). Although the rate of insertion and processing of the pro-OmpA EE was greatly stimulated by the electrochemical potential in uitro, the mutations clearly had deleterious effects on the rate compared to the wild-type pro- OmpA (compare the rates of processing of wild-type and pro- OmpA EE in the absence of NADH in Fig. 3, A and C). Other

types of interactions, unrelated to the effects of the electro- chemical potential, between the amino terminus of the mature domain of pro-OmpA and the secretory apparatus likely play important and perhaps more dominant roles than an electro- phoretic effect. Presumably, this may also include the diffi- culty in moving either negatively or positively charged resi- dues through the apolar lipid bilayer. Thus, on balance, it appears that a combination of the membrane electrochemical potential and the difficulty of penetrating the membrane with charged residues affects initiation of protein export.

In summary, the data presented here indicate that charged residues after the leader peptide affect the rate of the initial insertion, presumed loop formation, and cleavage of the pre- cursor in a electrochemical potential-dependent manner. The data strongly suggest that this is due to the A$ acting electro- phoretically.

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