Eur. J. Biochem. 124, 561 -566 (1982) 0 FEES 1982

Maturation of Exported Proteins in Escherichia coli Requirement for Energy, Site and Kinetics of Processing

j

Jean Marie PAGES and Claude LAZDUNSKI

Centre de Biochimie et de Biologie Moleculaire du Centre National de la Recherche Scientifique, Marseille

(Received November 9, 1981/January 18, 1982)

Precursor forms of periplasmic and outer membrane proteins were accumulated in phenethyl-alcohol- treated cells in membrane fractions. After removal of phenethyl alcohol, maturation occurred in the absence but not in the presence of carbonylcyanide rn-chlorophenylhydrazone (30 pM). The site and kinetics of processing were investigated for OmpA, LamB and OmpF proteins and for maltose binding protein and TEM p-lactamase. With regard to sites of processing, no fundamental difference between outer membrane and periplasmic proteins was observed. For maltose binding protein and TEM p-lactamase, maturation, like that of outer membrane protein precursors, occurred in membrane fractions. Processing of pro-OmpA protein was about as fast as that of pro- LamB protein whereas pro-OmpF protein appeared to mature more slowly. While carbonylcyanide m-chloro- phenylhydrazone (30 pM) prevented processing of all precursor forms, arsenate, which alters formation of ATP even when it was used at 1 mM, did not totally prevent maturation occurring. These results are discussed with regard to the biosynthesis and assembly of exported proteins.

Periplasmic and outer membrane proteins of Escherichia coli are produced in membrane-bound polysomes [I -31 in precursor form. Short-lived peptide extensions, called signal peptides [4], in these precursors are required for the initial attachment of the nascent polypeptide chains, carried by ribosomes, to the cytoplasmic membrane, as demonstrated through genetic studies [5]. These signal peptides, of char- acteristic structure [6], are usually processed during or shortly after synthesis of nascent proteins [7]. However, the transfer step itself across the membrane does not require processing [8,9].

In previous studies we have demonstrated that procaine, a local anesthetic, could inhibit the processing of various ex- ported protein precursor forms [lo, 11 ]. It was later shown that phenethyl alcohol, another anesthetic, had the same effect for outer membrane [I21 and periplasmic [I31 proteins.

In the present study, we have taken advantage of this technique which allows precursor forms of exported proteins to be accumulated in cells treated with these anesthetics. We have then asked whether the electrochemical gradient in the cytoplasmic membrane was required for processing, where did this processing occur and what were the kinetics of processing for various precursor forms of exported proteins?

Concomitantly with Enequist et al. [14], we have recently reported in a short note [I51 that energy was required for maturation of exported proteins. We have now extended this work in order to provide better answers to the various questions. The results, presented in this study, confirm that the electrochemical gradient across the membrane and not high-energy phosphate is required for maturation of peri- plasmic and outer membrane protein precursor forms, The site of processing could only be operationally defined since,

Enzymes. fi-D-Gahctosidase (EC 3.2.1.23); TEMI 8-lactamase or penicillin amino-/-lactamhydrolase (EC 3.5.2.6).

like other [16], we have encountered difficulties in assigning a precise location through conventional techniques. The main site of processing both for precursor forms of periplasmic and outer membrane proteins was found to be a pellet fractions insoluble in Triton X-100. Kinetics of processing observed for the various precursor forms slightiy differ from one protein to the other.

MATERIALS AND METHODS

Chemicals

[35S]Methionine (890 Ci/mniol) was purchased from Amer- sham. Phenethyl alcohol and carbonylcyanide m-chloro- phenylhydrazone were obtained respectively from Fluka AG and Calbiochem.

Bacterial Strains and Growth Conditions

Strains JF 404 and C 600 pBR 322 were generous gifts from Drs J. Foulds and P. Kourilsky respectively. Cells were grown at 37°C in a Tris-based medium supplemented with 16 mM maltose as source of carbon and energy [17].

Conditions for Radiolahelling oj Prolcins

Cells were grown at 37 "C to a density of 2 x lo8 cells/ml, they were centrifuged and resuspended in 1/20 of the original volume in fresh medium. After 15 min at 37"C, the culture was divided, a small aliquot was kept without addition as a control, in the other fraction 0.5 % (40 mM) phenetyl alcohol was added. After 3 min [35S]methion~ne (60 pCi/ml, 60 nm) was added in each fraction and the incubation was carried on for 15 min at 37 "C. Control cells were then centrifuged and

562

the pellet was frozen. Phenethyl-alcohol-treated cells were divided into two fractions, in one fraction carbonylcyanide m-chlorophenylhydrazone (CCCP 30 pM) was added and both fractions were rapidly centrifuged. The first pellet was resuspended in fresh medium to the original volume, and un- coupler-treated cells were resuspended similarly except that 30 pM carbonylcyanide m-chlorophenylhydrazone was ad- ded. Unlabelled methionine (70 mM) was added in both fractions and during the incubation at 37 "C, samples were removed at various times, centrifuged 1 min at 13 000 x g and cell pellets were immediately frozen in liquid nitrogen.

The extent of lysis during pulse and chase experiments was calculated by assaying P-galactosidase (induced by 1 mM isopropyl P-D-thiogalactoside). The lysis was never higher than 5 - 7 :<.

Prepurntion of Memhranr and Soluble Fructions

The cells pellets were resuspended in 50mM Tris pH 8.5, 2 mM EDTA with deoxyribonuclease I0 pgjml (DNase) and sonicated by five runs of 15 s in a Branson sonifier (model SIIO). A centrifugation at 12000 x g eliminated intact cells; the supernatant was then centrifuged at 140 000 x g and 4 "C for 45 min. Supernatant S1 represented the soluble material; pellet PI was treated with a buffer containing2 "/,TritonX-l00, 10 mM Hepes pH 7.4, 5 mM MgCl2 for 20 rnin at 2 0 ' C ac- cording to Schnaitman [18]. The extract mixture was cen- trifuged at 140000xg and 4°C for 45 min. Supernatant S2 was called the Triton-soluble fraction. Pellet P2 was solu- bilized in 2 sodium dodecyl sulfate for 10 min at 100 'C and was then centrifuged for 30 min at 13000 x g. Supernatant S3 was called sodium-dodecyl-sulfate-soluble fraction.

-LABELLED CELLS

1

1 S-o n

(Tris-EDTA-DNAse)

Centr i fugat ion 15 rnin 12000.q

-PELLET 1 SUPERNATANT - Centrifugation 45 min 140 0OO.g

I

SUPERNATANT - ' PELLET

Solubilizat ion

Triton-Hepes- MgCI, 20 rnin 20°C

Centr i fugat ion 45 rnin 140000.g 4

@ SUPERNATANT - PELLET

I Solu bilizat ion

SDS 2% IOmin 100°C

i Centr i fugat ion 30 min 13 0OO.g

PELLET SUPERNATANT

Fig. 1. Abbreviated nufline of the fractionation procedure used,for i,soluting the vurious cell compurtmtws

RESULTS Conditions of Immunoprecipitution

Efycct qj' Cell Treatment with Plienethyl Alcohol on the DiLytributio/l of' ProtpinLy Cell or membrane pellets were solubilized in 100 pl Of 2

sodium dodecyl sulfate at 100°C for 10 min and brought into a buffer containing 10 mM Tris, 150 mM NaC1, 0.05% methionine, 5 mM EDTA, 1 "/, Triton X-100, 0.2% sodium dodecyl sulfate at pH 7.4. Samples were clarified by centrifu- gation for 30 min at 13000 x g and incubated for 45 rnin in the presence of 3 p1 of a decoinplemented antiserum directed against the protein to be immunoprecipitated. An activated suspension (20 1.11) of Staphylococcus uureus cells (coated with protein A according to the method of Kessler [19]) was added to the mixture and the incubation was continued for 45 min. Cells were then centrifuged for 30 min at 13 000 x g, pellets were resuspended in 100 pl of the incubation buffer and applied on a 1 M sucrose cushion (1 ml). After centrifugation, pellets were washed twice with a buffer similar to the incubation buffer except that Triton X-100 was 0.1 instead of 1 and sodium dodecyl sulfate was 0.02 instead of 0.2 %. After the last washing with water, pellets were dried and taken up in the denaturation buffer used to apply samples on sodium dodecyl sulfate/polyacrylamide gels.

Sodium Dodecyl Sulfate Gel Electrophoresis and Fluorograpliy

Immunoprecipitated proteins were analysed by sodium dodecyl sulfate gel electrophoresis in 11 "/, polyacrylamide gels according to Lugtenberg et al. [20]. The gels were impregnated and fluorographed as described [22 1. Densitometer scannings of fluorograrns were carried out in a Vernon densitometer.

Detcc,een tfie b'ariou.~ Cell Compartments

Cell proteins were labelled as described above in the presence of phenylethyl alcohol and chased in absence or in presence of carbonylcyanide m-chlorophenylhydrazone. The repartition of radioactive proteins into various cell compart- ments was investigated in samples removed at various times during the chase. The outline of the fractionation procedure is described in Fig. 1. The supernatant S1 of the 140000 x g centrifugation contains periplasmic proteins, cytoplasmic proteins and inner membrane proteins which remain in this fraction when cells are disrupted by sonication, as previously found by Crowlesmith et al. [22]. This explains why very little protein, about 5%, was found to be solubilized from pellet PI by Triton X-100. Proteins solubilized from pellet P2 by sodium dodecyl sulfate probably belong mostly to the outer membrane since the protein pattern of the S3 fraction was found to be very similar to the characteristic outer mem- brane protein pattern (data not shown). As shown in Fig.2 the amount of radioactive proteins associated with the 140000 x g supernatant SI increased during the chase in the absence of phenethyl alcohol to reach a value close to that of control cells. This increase was achieved at the expense of pellet P1 that decreased to the control level. In contrast, in cells treated with carbonylcyanide m-chlorophenylhydrazone (30 pM) there was little variation in fractions S1 and PI. This result first suggested that precursor forms of exported proteins were mostly accumulated in fraction PI.

563

Fig.2. Distribution qf bheilidproteins in various /'kx,tions. Cells were labelled with [35S]methionine (60 pCi/ml. 60 nM) during 15 min at 37 'C in the presence of 0.5 ",; phenethyl alcohol and they were chased in the absence (A) or in the presence (B) ofcarbonylcyanide rn-chlol-ciphenylhydrazone (CCCP, 30 pM). A small aliquot was kept without addition of phenethyl alcohol as a control (C). Samples were removed at variou\ times (as indicated), frac- tionated according to the procedure described in Fig. 1. Radiolabelled proteins precipitated by trichloroacetic acid were dcrcrminated in each fraction and are presented as a percentage of total proteins. SI. SZ, S3, PI , P3 stand for supernatants and pellets obtained as described in Fig. 1

A

Effert ~f the Dissipution of' the Proton-Motive Forcr on the Processing o f Prcwrsar Fornzs qf Outer. Membrane Proteins

OnipA Protein. The precursor form of the OmpA protein was accumulated in the presence of phenethyl alcohol as previously described [12]. When cells were directly solubilized in 2 y,; sodium dodecyl sulfate without fractionation, practi- cally no processing of this precursor form was observed in the presence of carbonylcyanide m-chlorophenylhydrazone (30 FM) (data not shown). However, when cells were frac- tionated as described in Fig.1 and the OmpA precursor immunoprecipitated from supernatant fraction S3, even at the time 20 s during the chase, some processing had already occurred (Fig. 3A). In the presence of the uncoupler (Fig. 3 B) although the processing was significantly inhibited it could not be totally avoided. A clear difference between the chase in the presence or absence of the uncoupler could still be observed by comparing densitometer scannings of fluorograms (Fig. 3).

LamB Protein. The LamB protein, the lambda phage receptor [23], becomes a major protein of the outer membrane in cells grown in the presence of maltose [24]. The same analysis as that described for OmpA protein was applied. Again, very little processing was observed in the presence of uncoupler when immunoprecipitation was carried out on sodium dodecyl sulfate extracts of whole cells (data not shown). After fractionation, the precursor form was immuno- precipitated from the supernatant fraction SJ, as described above. We observed also that the processing was inhibited, although not totally, in the presence of carbonylcyanide m-chlorophenylhydrazone (30 pM) (Fig. 4A, C). When im- munoprecipitation was carried out on fraction Sz that should normally contain the inner membrane protein, about the same pattern as observed above for fraction S3 was obtained (Fig.4B, D). Since mature LamB protein should not be found in this fraction, this suggested that the Triton X-100 solubili- zation technique was not working in phenethyl-alcohol- treated cells. The kinetics of processing were investigated in the presence or absence of the uncoupler (data not shown). From this result the half-life of the precursor was found to be about 70 s under the conditions used.

8

Migration - Fig. 3. 6ffec.t oftlie dissipution of tlleproton-itrotrveforc.e on thrproce.wing of' the OmpA protein precursor. Cells, pulse-labelled and chased as de- scribed in Materials and Methods, were treated as indicated in Fig.l. OmpA protein was then immunoprecipitated from the sodium-dodecyl- sulfate-soluble fraction S1. Chase in the ahsence (A ) or presence (B) of carbonylcyanide rn-chlorophenylhydrazone (30 pM). Samples were removed a t 20 s (lane 2). I min (lane 3 ) , 2 rnin (lane 4), 20 min (lane 5), 60 min (lane 6). In lane 1 ( A and B) mature OinpA protein immunopreci- pitated from control cells was applied

564 A

B

C

D Migra t ion -

Fig. 4. Effect of the dissipation oj' the proton-motive force on the processing of the LamB protein precursor form. Cells, pulse-labelled and chased as described in Materials and Methods, were treated as indicated in Fig. 1. LamB protein was then immunoprecipitated from the sodium-dodecyl-sulfate- soluble fraction SS (A and C). The chase was carried out in the absence (A, B) or presence (C, D) of carbonylcyanide m-chlorophenylhydrazone (30 FM). LamB protein was also immunoprecipitated from the Triton-soluble fraction S2 (B and D). The times of chase were respectively 20 s (lane 2), 1 rnin (lane 3), 2 rnin (lane 4), 10 rnin (lane 5), 60 rnin (lane 6). In lane 1 (A, B, C and D) mature LamB protein, immunoprecipitated from control cells, was applied

A

B

Migrat ion - Fig. 5. Effect ofthe dissipation of the proton-motive force on the maturation of OmpF protein precursor form. Cells, pulse-labelled and chased as de- scribed in Materials and Methods, were treated as indicated in Fig. 1. OmpF protein was then immunoprecipitated from the sodium dodecyl sulfate soluble fraction SS. The chase was carried out in the absence (A) or presence (B) of carbonylcyanide m-chlorophenylhydrazone. The times of chase were respectively 20 s (lane 2), 1 min (lane 3), 2 min (lane 4), 10 min (lane 5 ) , 60 min (lane 6). In lane 1 (A and B) mature OmpF protein, immunoprecipitated from control cells, was applied

OmpF Protein. OmpF protein, also called matrix protein [25], is one of the major proteins of the outer membrane in Escherichia coli K12. An analysis similar to that described above indicated that the precursor form was also recovered from the supernatant fraction S3 and that the uncoupler again inhibited processing (Fig. 5A, B). This is better shown in the densitometer scannings of the fluorograms (Fig. 5) . Processing of OmpF protein appeared to be slower than that of OmpA and LamB proteins. Again processing of OmpF precursor form was almost absent in sodium dodecyl sulfate extracts of whole cells chased in presence of uncoupler (data not shown).

Eifect of the Dissipation of the Protein-Motive Force on the Processing of Precursor Forms of Periplasmic Proteins

Maltose binding protein is a periplasmic protein involved in maltose transport [26,27]. It is produced in large amounts in maltose-induced cells. The same analysis as that used for outer membrane proteins was applied. Again immuno- precipitations were carried out in sodium dodecyl sulfate extracts of whole cells pulsed and chased with methionine in the presence or absence of uncoupler. Virtually complete processing of the precursor form of maltose binding protein occurred during the chase in the absence of uncoupler but not in its presence (data not shown). Since mature maltose binding protein is soluble, we considered whether the pre- cursor form was also found in the supernatant fraction S3, as observed above for outer membrane protein precursor forms. This was indeed the case and the amount of precursor form precipitated from this fraction decreased during the chase (Fig. 6A). Concomitantly, the mature forin appeared in the 140000xg supernatant SI (Fig.6B). In contrast, in the presence of uncoupler, the precursor form remained asso- ciated to the supernatant fraction S3 (Fig.6C) and thus very little mature protein appeared in supernatant S1 (Fig. 6 D). This indicated that processing could occur in the absence of un- coupler but not in its presence. As for LamB protein, proces- sing was also found to occur in the Triton-soluble fraction Sz (data not shown).

A 565

1 2 3 4 5

Migration- C

1 2 3 4 5

Migration -

D Migration -

M m

Migration - Fig. 6. Effct o f f h e dissipation of'lhe proton-mofive.force on the processing of multose hindingprotein precursorform Cells, pulse-labelled and chased as described in Materials and Methods, were treated as indicated in Fig. 1. Maltose binding protein was then immunoprecipitated from fractions S3 (A, C) and SI (B, D). Chase in the absence (A, B) or presence (C, D) of carhonylcyanide m-chlorophenylhydrazone (30 p M ) . Samples were rernovcd at 20 s (lane 1 , A and C ; lane 2, B and D), 3 min (lane 2, A and C; lane 3, B and D), 2 min (lane 3, A and C ; lane 4, B and I ) ) , 10 min (lane 4. A and C ; lane 5 , B and D), 60 min (lane 5, A and C; lane 6, B and D). Mature maltose binding protein, immunoprecipitated from control cells, was applied in lane 1 (B and D)

Similar features were observed for the periplasmic TEM fl-lactamase of which the precursor form is processed post- translationally [7,28]; the only difference was that the mature form of TEM b-lactamase was released more slowly than that of maltose binding protein, from fraction Sz (data not shown).

Arsenate is a cell poison that interferes with formation of ATP but which does not prevent development of a potential gradient across the inner membrane [29]. The same experi- mental approach as that described above was used in phenethyl-alcohol-treated cells pulsed with [35S]methionine and chased in the presence or absence of arsenate. Even when it was used at high concentration (1 mM), this compound caused only a 70 inhibition in the rate of protein synthesis under conditions of recovery from phenethyl alcohol treat- ment. Under these conditions, we found a partial inhibition of processing of pro-LamB protein (data not shown) instead of a complete inhibition as observed in the presence of car- bonylcyanide m-chlorophenylhydrazone (Fig. 4C, lane 6). However, we cannot use this data to conclude that only the electrochemical gradient across the membrane is required for processing of precursor forms of exported proteins because a high residual protein synthesis (30 %) remains after arsenate treatment which suggests only a partial depletion of ATP levels.

DISCUSSION

We had previously demonstrated [13] that phenethyl- alcohol causes extensive alterations in the localization of proteins in Escherichia di. Cell fractionation was carried out

in this previous study by converting cells to spheroplasts. Through this technique, it was determined that the bulk protein content of the cytoplasmic membrane was increased in the presence of phenethyl alcohol which prevents processing of precursor forms of outer membrane [I21 and periplasmic [13] proteins. This result, as well as other studies [12,22], strongly suggested that precursor forms of exported proteins were accumulated in the inner membrane. In this study too many samples had to be handled at the same time and the fractionation procedure had to be simplified as shown in Fig. 1. In the technique used, cells were first disrupted soni- cation and as a consequence most of the inner membrane (Triton-soluble fraction [181) was not sedimented after centrifugation for 30 min at 140000 x g [22] and thus was recovered in supernatant S1. Therefore. one could expect to find precursor forms in this fraction Sc. In fact, they were recovered both from pellet PZ after sodium dodecyl sulfate solubilization and from the Triton-soluble fraction Sz. Maturation of both outer membrane and periplasmic pro- teins occurred in fractions & and Sz. However, the fact that mature LamB protein was found in fraction S2 (Fig.4) strongly suggests that specific solubilization of the inner membrane proteins by Triton X-100 [IS] did not work in phenethyl-alcohol-treated cells. Therefore, on can only con- clude that both precursors of periplasmic and outer membrane proteins accumulate and are processed in membrane fractions.

It is worth pointing out that during the sonication and fractionation procedures some precursor processing, not ob- served when whole cells were directly solubilized in sodium dodecyl sulfate, occurred.

566

The kinetics of processing were similar for OmpA and LamB proteins, but appeared to be slightly slower for OmpF protein (Fig. 3 - 5).This result is in agreement with previously published data [22,30]. The apparent half-life of the pro- LamB protein was estimated to be about 70 s. Since the estimation at 25 "C for the pro-OmpA protein was 30 s [22], this suggests that processing may be significantly slower in phenethyl-alcohol-treated cells.

The finding of precursor forms of maltose binding protein and of p-lactamase, two periplasmic proteins, in the S3 fraction in addition to SZ was quite a surprise. There was no doubt that they were there because of the presence of the signal peptides since cleavage of these peptides, was ac- companied by release from this S3 fraction. The mature pro- teins released were then recovered in the S1 supernatant which comprises periplasmic proteins. Furthermore, the inhibition of cleavage in the presence of uncoupler prevented the release of precursor forms from their compartment (S3 fraction). Thus, only the signal peptide (comprising 26 and 23 amino-acid residues for maltose binding protein [31] and p-lactamase [32] respectively) is responsible for segregation of these proteins in a cell fraction that exclusively contains membrane pro- teins. Some precursor forms of exported proteins were im- munoprecipitated from the S2 fraction. However, in contrast to the results obtained after immunoprecipitation from the SB fraction some mature protein was always present in this fraction, even at early times of chase.

To conclude, using completely different techniques both Enequist et al. [I41 and our own studies suggest that an electro- chemical gradient across the cytoplasmic membrane is required for processing of exported proteins. It is not really surprising that energization of the membrane has an effect on various processes occuring in this locus. Indeed, assuming a potential of 100 mV [33] for a membrane 7.5 nm thick, this corresponds to about 13 kV/cm. The application of such high fields to biological membranes has been shown to cause various effects [34]. It is probably too early to speculate on the nature of the effect on processing. However, since it has been de- monstrated here that precursors, accumulated in the mem- brane fraction, remain there in the absence of membrane potential, it is possible that their orientation within the mem- brane, rather than their location, is altered. With regard to this problem, the case of exported proteins would therefore be similar to that of the MI3 coat protein [35]. For this protein uncouplers prevent it both from being processed and from integrating into a transmembrane conformation [36].

Further studies are now in progress to determine precisely why the electrochemical gradient across the cytoplasmic membrane is required for maturation of exported proteins in E. coli.

Nore. After this paper was submitted, results similar to those presented here, but dealing with the periplasmic leucine-specific binding protein of E. coli, were reported [37].

We thank Drs Szmelcman, Schwartz, Rosenhusch, Cesareni and Lugtenherg for generous gifts of antisera. This work was supported by

the Institut National de la SantP et de la Recherche Midicule (contract 79.4.202.1) and Action Thimatiques P rogrummPs Microbiology.

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J . M. Pages and C. Lazdunski, Centre de Biochimie et de Biologie Moleculaire du Centre National de la Recherche Scientifique, 31 Chemin Joseph-Aiguier, F-I 3277 Marseille-Cedex-9, France