Co-translocation of a Periplasmic Enzyme Complex by a Hitchhiker Mechanism through the Bacterial Tat Pathway*

Agnès RodrigueDagger , Angélique ChanalDagger , Konstanze Beck§, Matthias Müller§, and Long-Fei WuDagger parallel

From the Dagger  Laboratoire de Chimie Bactérienne, UPR9043 CNRS, Institut de Biologie Structurale et Microbiologie, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France and the § Institut für Biochemie und Molekularbiologie and  Fakulatät für Biologie, Universität Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial periplasmic nickel-containing hydrogenases are composed of a small subunit containing a twin-arginine signal sequence and a large subunit devoid of an export signal. To understand how the large subunit is translocated into the periplasm, we cloned the hyb operon encoding the hydrogenase 2 of Escherichia coli, constructed a deletion mutant, and studied the mechanism of translocation of hydrogenase 2. The small subunit (HybO) or the large subunit (HybC) accumulated in the cytoplasm as a precursor when either of them was expressed in the absence of the other subunit. Therefore, contrary to most classical secretory proteins, the signal sequence of the small subunit itself is not sufficient for membrane targeting and translocation if the large subunit is missing. On the other hand, the small subunit was required not only for membrane targeting of the large subunit, but also for the acquisition of nickel by the large subunit. Most interestingly, the signal sequence of the small subunit determines whether the large subunit follows the Sec or the twin-arginine translocation pathway. Taken together, these results provide for the first time compelling evidence for a naturally occurring hitchhiker co-translocation mechanism in bacteria.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteins destined for secretion, membrane integration, or assembly into organelles are sorted with high fidelity to their respective intracellular sites by virtue of targeting signals encoded within the primary structures of the nascent polypeptides themselves. The principal role of targeting signals is to mediate the engagement of the exported protein with components of the specific translocation machinery (1-3). In these cases, signal sequences are specifically recognized by a cytosolic chaperone or a targeting factor and act as true targeting signals. Alternatively, the function of signal sequences is proposed as being to delay the folding of the mature portion of an exported protein, allowing binding of an export-specific chaperone to its unfolded mature portion and thereby keeping the exported protein in a translocation-competent configuration (4).

Hydrogenases are omnipresent in bacteria and archaea (5). They catalyze the reversible oxidation of hydrogen and allow bacteria to use hydrogen as an energy source for their growth. Hydrogenases can be divided into two major superfamilies: (a) nickel-iron hydrogenases (NiFe hydrogenases), and (b) iron-only hydrogenases (Fe hydrogenases). They are generally composed of a small subunit of about 30 kDa and a large subunit of 60 kDa. All small subunits of periplasmic or membrane-bound hydrogenases contain an N-terminal signal sequence possessing a conserved twin-arginine motif, which is removed once the hydrogenases are translocated into the periplasm (5, 6). The large subunits of NiFe hydrogenases show no N-terminal processing, but they possess a C-terminal extension sequence composed of one to two dozen residues. The extension sequence seems to keep the precursor of the large subunit in a conformation competent for nickel acquisition, and it is removed by a specific cytoplasmic protease upon nickel incorporation (7). The large subunits, therefore, are devoid of any known signal sequence required for the export of proteins. They are assumed to be co-translocated with the small subunits (5, 6).

The small and large subunits of hydrogenase 2 (HYD2)1 of Escherichia coli are encoded by hybO and hybC of the hybOABCDEFG operon, respectively (8, 9). HYD2 is an extrinsic membranous protein that is attached to the periplasmic side of the cytoplasmic membrane by a 5-kDa fragment of its small subunit (10, 11). An active HYD2 can be released from spheroplasts by limited trypsin proteolysis (11). The acquisition of nickel in the cytoplasm is a prerequisite for HYD2 export (11), which is mediated by the twin-arginine translocation (Tat) pathway (12, 13). In this communication, we show that there is an interdependence between the small and the large subunits for their export and that the signal sequence of the small subunit determines the type of export pathway chosen by the large subunit. We thus provide the first example of a naturally occurring hitchhiker co-translocation of a dimeric enzyme across the bacterial cytoplasmic membrane.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains, Plasmids, and Growth Conditions-- Bacterial strains and plasmids used in this study are listed in Table I. To construct the Delta hybOABC mutant, the 3421-base pair XhoI-BglII fragment containing hybOABC was replaced by the promoterless cassette encoding beta -glucuronidase and KanR that was obtained by SalI-BglII digestion of the plasmid pUIDK2 (14). The transcriptional hybO-uidA fusion was recombined back to the chromosome of recD strain D355 (15) and was further moved into strains MC4100, B834, and HYD720 via P1cml transduction (16). Similarly, the secAts and secYts mutations were introduced into various strains by P1-mediated transduction, selection for tetracycline resistance, and screening for thermosensitivity.

                              
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Table I
Bacterial strains, lambda, and plasmids

The bacteria were routinely grown in LB medium, on LB plates, or in minimal M9 medium as described previously (11, 17, 18).

Preparation of Subcellular Fractions and Enzyme Assays-- Periplasm, spheroplasts, membrane, and cytoplasmic fractions were prepared by lysozyme/EDTA/cold osmoshock and ultracentrifugation, as described previously (11, 18). To extract peripherally bound membrane proteins, the membrane was washed with 6 M urea or 100 mM sodium carbonate (pH 10). To further separate membrane proteins from aggregates, the membrane fractions were solubilized by 4% Triton X-100 in 40 mM Tris-HCl (pH 7.6) and centrifuged using an Airfuge at 30 p.s.i. for 10 min. To release HYD2 from the washed spheroplasts, limited trypsin digestion was performed as described previously (11).

Hydrogenase activity was measured by following the H2-linked reduction of benzyl viologen spectrophotometrically at 600 nm or by activity staining as described previously (16, 17).

Immunological Procedures and in Vivo and in Vitro Specific Labeling of hyb Gene Products-- Immunoblotting was performed by using the ECL method according to the manufacturer's instructions (Amersham Corp.). To prepare antiserum against HybO, the hybO gene was cloned into pET22b+, and 6-His was added at the C terminus of the HybO. The recombinant HybO6His was solubilized by guanidine hydrochloride from inclusion bodies and partially purified on a nickel nitrilotriacetic acid column according to the manufacturer's instructions (Qiagen). Gel slices containing HybO6His were used in standard immunization protocols for rabbits (Eurogentec). The resulting antiserum also contains antibodies that recognize contaminating antigens. Nonspecific antibodies were removed by absorption to an acetone powder prepared with whole cells of ENF1 (Delta hybOABC) as described in Ref. 19.

The hyb gene products were specifically labeled by [35S]methionine in vivo using T-7 RNA polymerase (20) or in vitro using a plasmid-directed transcription-translation system (21).

In Vitro Cross-Linking-- In vitro cross-linking with formaldehyde was performed as described in Ref. 22, with modifications. Soluble S-135 cytoplasmic fractions prepared from the strains ENF1/pHyb84 (Delta hybOABC/hyb(A-G)+) and ENF1/pHyb411 (Delta hybOABC/hybO+) were subjected, separately or as a mixture, to treatment with 0.1% formaldehyde. The reaction was incubated at room temperature for 30 min. Aliquots were removed, and the reaction was stopped by the addition of 50 mM Tris-HCl (pH 7.6) and benzonase. To dissociate cross-linked complexes, samples were heated at 100 °C for 15 min, whereas the control was kept at 37 °C. All samples were treated at 90 °C for 5 min before applying them to the gel.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of the hyb Operon and Construction of the Delta hybOABC Mutant-- To study the translocation mechanism, we cloned the hyb operon by using the Kohara collection (23). Among five cosmids covering the 65 min region of the E. coli chromosome, only lambda 5C10 was able to confer HYD2 activity on mutant HDJ123 that is pleiotropically defective in hydrogenase activities (Delta hya, Delta hybBC, and Delta hyc). A 15-kilobase BamHI fragment containing the entire hyb operon was obtained from lambda 5C10 and cloned into pUC18, resulting in the plasmid pHyb11. A chromosomal deletion mutant in which the hybOABC genes were replaced by a uidA-KanR cassette was then constructed as described under "Experimental Procedures." As expected, the resulting mutant ENF1 was deficient in HYD2 activity and was devoid of both the small (HybO) and the large (HybC) subunits (data not shown). The wild type phenotype was completely restored to mutant ENF1 by the introduction of plasmid pHyb11 harboring the entire hyb operon.

Influence of the Large Subunit on the Targeting of the Small Subunit-- In cells expressing the entire hyb operon, two forms of HybO, the small subunit of HYD2, were detected. As expected, the larger precursor form was found in the cytoplasm (Fig. 1A, lane 1), whereas the mature form, with the signal sequence removed, was recovered from the membrane fraction (Fig. 1A, lane 2). Interestingly, when it was synthesized alone, HybO accumulated as a precursor (HybO-p) in the cytoplasm and was completely absent from the membrane (Fig. 1A, lanes 3 and 4). Therefore, unlike most classical secretory proteins, the signal sequence of the small subunit of HYD2 itself is not sufficient for membrane targeting and translocation.


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Fig. 1.   Influence of HybC on the membrane targeting of HybO. Crude extracts (E), cytoplasmic fraction (C), membrane fraction (M), Triton X-100-solubilized membrane (S), and the insoluble pellet (P) were prepared from ENF1 (Delta hybOABC; panel A) carrying plasmids pHyb14 (hyb(O-G)+), pHyb411 (hybO+), or pHyb41 (hyb(O-B,D-G)+) or from NH1 (Delta hybOABC-Delta nik; panel B) containing plasmid pHyb14 grown without (-NiCl2) or with (+NiCl2) 0.3 mM NiCl2 and separated on a 12.5% SDS-gel. Precursor (HybO-p) and mature HybO (HybO-m) were detected by antiserum against HybO and are indicated on the right. Plasmid pHyb41 contains a 543-base pair in-frame deletion in hybC and, as verified in an in vivo labeling system, directs the biosynthesis of a truncated 42-kDa large subunit.

We investigated whether the large subunit (HybC) was necessary for the export of the small subunit. In the presence of a truncated HybC (see the Fig. 1 legend), the small subunit accumulated as a precursor in the cytoplasm (Fig. 1A, lane 5). Under these conditions, the precursor was also detected in a bona fide membrane pellet (Fig. 1A, lane 6). Further analysis, however, revealed that the pelleted precursor of the small subunit reflected aggregated material (Fig. 1A, lane 8) because it was absent from the Triton X-100-solubilized membrane fraction (Fig. 1A, lane 7). As a consequence, truncation of the large subunit resulted in the formation of aggregates of the small subunit.

We previously showed that in the nik mutant, which is deficient in the specific nickel transport system, the large subunit of HYD2 accumulates as a non-processed precursor in the cytoplasm, but the addition of nickel to the growth medium restores processing of the large subunit and its membrane targeting (11, 17). In a double Delta nik-Delta hybOABC mutant complemented with the entire hyb operon ((hybO-G)+) and grown in the absence of nickel, the small subunit HybO was detected as HybO-p in crude extracts and the cytoplasm, but it was completely absent from the membrane (Fig. 1B, lanes 2, 3, and 1, respectively). HybO-p synthesized under this condition was very labile, and a slightly smaller breakdown product of HybO was observed in this strain. The addition of nickel to the growth medium resulted in membrane targeting and maturation of the precursor of the small subunit (Fig. 1B, lanes 5 and 6), implying a successful translocation of HybO and the removal of its signal sequence. These results indicate that a deficiency in nickel incorporation and in large subunit processing directly or indirectly affects the targeting and translocation of the small subunit of HYD2.

Membrane Targeting and Processing of the Large Subunit of HYD2-- Because the large subunits of hydrogenases are devoid of signal sequences, they are assumed to be co-translocated with the small subunits. We therefore analyzed the effect of a complete depletion of hybO on the translocation of the large subunit using immunoblot analysis. In the presence of the small subunit, the processed form (HybC-pf) of the large subunit was detected in both the membrane and the cytoplasm (Fig. 2, lanes 2 and 4). The non-processed precursor (HybC-np) was present exclusively in the cytoplasm (Fig. 2, lane 4). This result indicates that only the processed form of the large subunit is efficiently targeted to the membrane. In addition, the large subunit was successfully translocated into the periplasm because active HYD2 was released from spheroplasts by treatment with trypsin (data not shown). On the other hand, in the absence of the small subunit, the large subunit accumulated exclusively as a non-processed precursor (Fig. 2, lane 5), it was absent from the membranes (Fig. 2, lane 1), and it was totally localized in the cytoplasm (Fig. 2, lane 3). These findings imply a failure of nickel incorporation into the large subunit, which is the normal prerequisite for its processing. Therefore, the small subunit is required not only for membrane targeting, but also for the processing of the large subunit.


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Fig. 2.   Requirement of the small subunit for membrane targeting of the large subunit. Crude extracts (E), cytoplasmic fraction (C), and membrane fraction (M) were prepared from ENF1/pHyb14 (hyb(O-G)+) and ENF1/pHyb84 (hyb(A-G)+) and separated on a 7.5% SDS-gel. The presence (+) or absence (-) of the hybO gene product in corresponding extracts is indicated. The nonspecific contaminating band (NS), non-processed precursor (HybC-np), and the processed form of HybC (HybC-pf) detected by immunoblot are indicated on the right. The increase in mobility of HybC-pf compared with the precursor HybC-np results from the removal of the C-terminal extension sequence of the precursor upon nickel incorporation (11).

Formation of a HybO-HybC Complex-- The interdependence between the two subunits for their translocation suggests a complex formation before export. We assessed this possibility using cross-linking and immunoblot analysis. Membrane-free S-135 fractions were prepared from spheroplasts of the mutant ENF1 (Delta hybOABC) complemented either by pHyb84 containing the hyb operon except hybO or by pHyb411 carrying only the hybO gene. The two extracts were then treated separately or as a mixture with formaldehyde. A cross-linking product of about 100 kDa detected by antisera against the small or the large subunits was obtained only if both the small and large subunits were present in the reaction medium (Fig. 3, lanes 4 and 5 compared with lanes 1, 2, 7, and 8). Moreover, as expected for a formaldehyde cross-linking product, this band disappeared when samples were heated to 100 °C (Fig. 3, lanes 3 and 6). These results suggest the formation of a HybO-HybC complex under this condition, e.g. in the absence of membranes. The low amount of the HybO-HybC complex obtained correlated with the low quantity of small subunit available in the extract (lane 2); HybO present in the S-135 fraction seems to be completely converted into the complex (lane 4 versus lane 2). However, release of the small subunit from the HybO-HybC complex by heating was not detected, probably because of a degradation or aggregation of HybO due to its labile and poorly soluble nature. The authenticity of HybO was established by comparing the S-135 fraction of ENF1/pHyb411 (hybO+; lane 2) with that of ENF1/pHyb84 (hybO-; lane 1) and was independently confirmed by in vitro transcription/translation using various plasmids (data not shown). In contrast, the 92- and 60-kDa bands are contaminating bands that are not related to HybO because they were found even in the absence of the hybO gene (Fig. 3, lane 1).


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Fig. 3.   In vitro cross-linking of HybO and HybC. Membrane-free S-135 fractions were subjected to treatment with the cross-linker formaldehyde, as indicated under "Experimental Procedures," separated on a 10% denaturing SDS-polyacrylamide gel, and electrotransferred onto a polyvinylidene difluoride membrane. Half of the membrane was incubated with antiserum against HybO (Anti-HybO), and the other half was incubated with antiserum against HybC (Anti-HybC), and both were developed by the ECL chemiluminescence method (Amersham Corp.). The presence (+) or absence (-) of HybO or HybC in the cross-linking reaction is indicated at the bottom. The dissociation of cross-linked complexes was achieved by heating the samples at 100 °C (+), whereas the control was treated at 37 °C (-) before applying the samples to the gel. Polypeptides detected by antisera against HybO and HybC are indicated on the left and right, respectively.

Sec-independent Translocation of HYD2 through the Tat Pathway-- The Tat pathway, which is required for the translocation of proteins carrying a signal sequence with an essential twin-arginine motif, has been recently identified in E. coli (12, 13, 24-26). We previously reported that one of the substrates of the Tat pathway, the periplasmic trimethylamine N-oxyde reductase, is exported independently of the Sec machinery (18). The above results indicating a co-translocation of the large and the small subunit of HYD2 are inconsistent with a passage of HYD2 through the Sec machinery, which is believed to accommodate single, unfolded polypeptides. Indeed, the large subunits of HYD2 were found mainly in the membrane fraction of a secY mutant (Fig. 4, lanes 5 and 6), and HybC was released from spheroplasts by limited trypsin digestion (lane 8), indicating a normal translocation of HYD2 across the cytoplasmic membrane of the secY mutant. The correct phenotype of the secYcs mutant used was confirmed by the accumulation of the precursor of MalE (lane 2). On the contrary, HybC accumulated in the cytoplasm of the tatC mutant (lanes 3 and 4), and it was not accessible from the spheroplasts (lane 7), which confirms the previous observation that translocation of Hyd2 is dependent on components of the Tat pathway (12, 13).


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Fig. 4.   Sec-independent translocation of HYD2. Crude extracts (E), cytoplasmic fractions (C), membranes (M), or trypsin-released protein fractions (T) prepared from the secYcs (CU164) (lanes 2, 5, 6, and 8) or tatC (lanes 1, 3, 4, and 7) mutants were resolved by 13% (lanes 1 and 2) or 10% (lanes 3-8) SDS-polyacrylamide gel electrophoresis. MalE (lanes 1 and 2) and HybC (lanes 3-8) were detected by immunoblots. The precursor (MalE-p) and the mature form (MalE-m) of MalE and the large subunit (HybC) are indicated on the left and right, respectively.

The signal sequence of the small subunit determines the translocation pathway to which the large subunit is channeled-- The twin-arginine signal sequence of the small subunit is likely to be the determinant for directing HybC to the Tat apparatus. To address this point, we substituted the first 27 N-terminal residues of HybO, including the twin-arginine motif, with the 22 residues of the typical Sec-dependent signal sequence of PelB. The resulting plasmid, pHyb55, directed the biosynthesis of the chimera PelBssHybO, which accumulated as a 39-kDa precursor and ran between the precursor and mature form of HybO (Fig. 5A). Under this condition, the chimera and the large subunit were found mainly in the membrane fractions (Fig. 5B, lanes 3 and 4). However, they were absent from either the periplasm or the trypsin-solubilized fraction (data not shown), suggesting that they are targeted to but not translocated across the membrane. Nevertheless, the large subunit was tightly bound to the membrane (see below). It seems likely that the chimera forms a PelBssHybO-HybC complex that is targeted by virtue of the PelB signal sequence to the translocase SecYEG, and it would then become stuck in the translocation channel.


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Fig. 5.   Influence of the signal sequence of HybO on the choice of export pathway used by HybC. A, crude extracts of ENF1/pHyb411 (Delta hybOABC/hybO+; lane 1), ENF1/pHyb55 (Delta hybOABC/hyb(pelBssO-G)+; lane 2), and ENF1/pHyb14 (Delta hybOABC/hyb(O-G)+; lane 3) were separated on a 12.5% SDS-gel and analyzed by immunoblot using antiserum against HybO. The precursor (HybO-p) and mature form (HybO-m) of the small subunit and the precursor of the chimera (PelBssHybO-p) are indicated on the right. B, cells containing pHyb14 (hyb(O-G)+; lanes 1 and 2) or pHyb55 (hyb(pelBssO-G)+; lanes 3-8) were grown at 30 °C to early exponential phase, shifted to 42 °C, and incubated for an additional 3 h. Thirty µg each of proteins of cytoplasmic fractions (C) and membrane fractions (M) prepared from Delta hybOABC (lanes 3 and 4), secYts-Delta hybOABC (lanes 1, 2, 5, and 6), and secAts-Delta hybOABC (lanes 7 and 8) mutants were separated on a 10% SDS-gel and probed with antiserum against the small (B1) or large subunits (B2). To analyze both the small and large subunits on the same gel, a compromised concentration (10%) of polyacrylamide was used. This concentration is not appropriated for visualizing the tiny migration difference between HybO-m and PelBssHybO-p, as observed on a 12.5% gel in A. The small (HybO) or large (HybC) subunits and the nonspecific band are indicated on the right. C, crude extracts prepared from secYts (lane 1), wild type strain without plasmid (lane 2), or complemented by pHyb14 (lane 3) or pHyb55 (lane 4) grown at 30 °C were separated on 13% SDS-gels and analyzed by immunoblotting using antisera to MalE. The precursor (MalE-p) and processed MalE (MalE-m) are indicated on the right. D, membrane fractions were prepared from Delta hybOABC (lanes 1, 4, and 7), secYts-Delta hybOABC (lanes 2, 5, and 8), and secAts-Delta hybOABC (lanes 3, 6, and 9) complemented by pHyb55. Urea-extracted fractions (lanes 1-3), Triton X-100-solubilized membranes (T-X100; lanes 4-6), and insoluble fractions (lanes 7-9) were separated on a 7.5% SDS-gel and analyzed by immunoblot using antiserum against the large subunit.

This assumption was confirmed by the following observations. First, the doubling time during the growth of MC4100/pHyb55, which synthesizes the PelBssHybO-HybC complex, increased by 50% compared with that for MC4100/pHyb14 in which HybO is synthesized with its native twin-arginine signal sequence. Second, the precursor of MalE accumulated in MC4100/pHyb55 (Fig. 5C, lane 4) to the same extent as in the secY mutant (lane 1), but it was absent from MC4100/pHyb14 (lane 3). Therefore, the jamming of the Sec translocon by the PelBssHybO-HybC complex resulted in an inhibitory effect on bacterial growth and led to the accumulation of a precursor of a Sec substrate. Finally, in contrast to the native HybO-HybC complex that is located exclusively in the membrane of the secY mutant (Fig. 5, B1 and B2, lanes 1 and 2), targeting of the chimeric PelBssHybO-HybC complex was clearly affected by sec mutations. Thus, the large subunit HybC accumulated mainly in the cytoplasms of both secY and secA (Fig. 5B2, lanes 5 and 7), whereas the amount of PelBssHybO detected in these mutants was strongly reduced (Fig. 5B1, lanes 5-8), presumably due to a degradation of misfolded or non-protected material. The remainder of HybC found to co-sediment with membranes (lanes 6 and 8) was further analyzed as to the nature of its membrane association. HybC co-expressed with PelBssHybO was completely removed from secA membranes by 6 M urea (Fig. 5D, lane 3), whereas no HybC was extracted from the membranes of the wild type strain and the secY mutant (Fig. 5D, lanes 1 and 2). A second extraction of these membranes with sodium carbonate did not further remove HybC (data not shown). When the extracted membranes were treated with Triton X-100, only HybC in the wild type strain was solubilized (Fig. 5D, lane 4), whereas that in the secY mutant remained in the insoluble fraction (Fig. 5D, lane 8). Thus, in the presence of PelBssHybO, most HybC looses its membrane association upon inactivation of SecY and SecA, with residual sedimenting material being only loosely attached or aggregated. The combined results therefore indicate that HybC in a PelBssHybO-HybC complex is targeted to the membrane by the Sec machinery recognizing the PelB signal sequence of the chimeric small subunit. These findings strongly support the idea that HybO and HybC form a complex before translocation and that HybC is targeted to the membrane via a hitchhiker mechanism.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Most extracytoplasmic proteins are synthesized with a signal sequence that targets them for export. Removal of the signal sequence or mutations in the signal sequence considerably decrease the efficiency of protein export. However, it has been reported that polypeptides lacking a signal sequence can be effectively imported into the peroxisomal matrix in a piggyback fashion on other polypeptides containing signal sequences (27). Bacterial hydrogenases are composed of small subunits with the twin-arginine signal sequence and large subunits devoid of any export signal. A naturally occurring co-translocation between the subunits has been proposed (5, 6). In this study, we observe an interdependence between the small and the large subunits for the translocation of HYD2 across the cytoplasmic membrane. Most importantly, we show that the signal sequence of the small subunit determines the export pathway followed by the large subunit.

We considered two models consistent with a hitchhiker co-translocation mechanism. According to the first model, membrane targeting of the two subunits of HYD2 is a sequential event. The precursor of the small subunit is targeted alone to the membrane with the help of its signal sequence. The incorporation of nickel into the large subunit leads to the removal of its C-terminal extension sequence and results in a conformational change, which allow the processed large subunit to specifically interact with the membrane-bound small subunit. This interaction triggers the membrane insertion of the large subunit and the formation of the complex, which then crosses the membrane by an unknown mechanism. According to the second model, the small and the large subunits of HYD2 first form a complex, which is followed by processing of the large subunit and then by membrane targeting of the complex by virtue of the signal sequence of the small subunit. Our findings are more in favor of the second model. When the small subunit was expressed alone, it accumulated in the cytoplasm as a precursor. In addition, membrane targeting of the small subunit required not only the presence of the large subunit but also nickel incorporation into the large subunit and processing of the large subunit. Reciprocally, depletion of the small subunit prevented the large subunit from being targeted to the membrane and affected the incorporation of nickel into the large subunit and its processing. In addition, the substitution of the twin-arginine signal sequence of the small subunit with a Sec-dependent signal sequence inhibited nickel acquisition and processing of the large subunit (data not shown). Because the acquisition of nickel by the large subunit occurs in the cytoplasm and is a prerequisite for HYD2 translocation (11), the interdependence implies a direct contact between the subunits and strongly suggests the formation of a complex before translocation. Consistently, we observed that HybO and HybC can form a complex in vitro in the absence of membranes. Taken together, our results show that the bacterial Tat pathway is capable of translocating oligomeric complex across the cytoplasmic membrane in a piggyback fashion, and thus it shares mechanistic similarities with the pathway used in protein import into the peroxisomes, in addition to the characteristics common to the Sec-independent, Delta pH-driven import pathway of plant thylakoids.

    ACKNOWLEDGEMENTS

We are grateful to M. A. Mandrand-Berthelot, C. L. Santini, B. Ize, G. Giordano, and M. Chippaux for valuable discussion and for their constant interest in this work. We thank E. Bouveret for advice regarding the in vitro cross-linking experiment and T. Pamler, N. Budisa, A. Filloux, and D. Boxer for the gifts of bacterial strains and HYD2 antiserum used in this study.

    FOOTNOTES

* This work was supported by grants from Centre National de la Recherche Scientifique (to UPR9043 CNRS), by Procope Grant 95068 from Ministère des Affaires Etrangères (to L.-F. W.), by a grant from Deutscher Akademischer Austauschdienst (to K. B.), and by a fellowship from Fondation pour la Recherche Medicale (to A. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed. Tel.: 33-491-164-212; Fax: 33-491-718-914; E-mail: wu{at}ibsm.cnrs-mrs.fr.

    ABBREVIATIONS

The abbreviations used are: HYD2, hydrogenase 2; Tat, twin-arginine translocation.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Berks, B. C. (1996) Mol. Microbiol. 22, 393-404[Medline] [Order article via Infotrieve]
  2. Robinson, C., and Mant, A. (1997) Trends Plant Sci. 2, 431-437[CrossRef]
  3. Zheng, N., and Gierasch, L. M. (1996) Cell 86, 849-852[Medline] [Order article via Infotrieve]
  4. Randall, L. L., Topping, T. B., and Hardy, S. J. (1990) Science 248, 860-863[Medline] [Order article via Infotrieve]
  5. Wu, L.-F., and Mandrand, M. A. (1993) FEMS Microbiol. Rev. 10, 243-269[CrossRef][Medline] [Order article via Infotrieve]
  6. Voordouw, G. (1992) Adv. Inorg. Chem. 38, 397-422
  7. Binder, U., Maier, T., and Böck, A. (1996) Arch. Microbiol. 165, 69-72[CrossRef][Medline] [Order article via Infotrieve]
  8. Menon, N. K., Chatelus, C. Y., Dervartanian, M., Wendt, J., Shanmugam, K. T., Peck, H. D., Jr., and Przybyla, A. E. (1994) J. Bacteriol. 176, 4416-4423[Abstract]
  9. Sargent, F., Ballantine, S. P., Rugman, P. A., Palmer, T., and Boxer, D. H. (1998) Eur. J. Biochem. 255, 746-754[Abstract]
  10. Ballantine, S. P., and Boxer, D. H. (1986) Eur. J. Biochem. 156, 277-284[Abstract]
  11. Rodrigue, A., Boxer, D. H., Mandrand-Berthelot, M. A., and Wu, L.-F. (1996) FEBS Lett. 392, 81-86[CrossRef][Medline] [Order article via Infotrieve]
  12. Bogsch, E., Sargent, F., Stanley, N. R., Berks, B. C., Robinson, C., and Palmer, T. (1998) J. Biol. Chem. 273, 18003-18006[Abstract/Free Full Text]
  13. Sargent, F., Bogsch, E. G., Stanley, N. R., Wexler, M., Robinson, C., Berks, B. C., and Palmer, T. (1998) EMBO. J. 17, 3640-3650[Abstract/Free Full Text]
  14. Bardonnet, N., and Blanco, C. (1992) FEMS Microbiol. Lett. 72, 243-247[Medline] [Order article via Infotrieve]
  15. Russell, C. B., Thaler, D. S., and Dahlquist, F. W. (1989) J. Bacteriol. 171, 2609-2613[Medline] [Order article via Infotrieve]
  16. Wu, L.-F., and Mandrand-Berthelot, M. A. (1986) Biochimie (Paris) 68, 167-179[Medline] [Order article via Infotrieve]
  17. Rodrigue, A., Batia, N., Müller, M., Fayet, O., Böhm, R., Mandrand-Berthelot, M. A., and Wu, L.-F. (1996) J. Bacteriol. 178, 4453-4460[Abstract]
  18. Santini, C. L., Ize, B., Chanal, A., Müller, M., Giordano, G., and Wu, L.-F. (1998) EMBO J. 17, 101-112[Abstract/Free Full Text]
  19. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  20. Tabor, S., and Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1074-1078[Abstract]
  21. Müller, M., and Blobel, G. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7421-7425[Abstract]
  22. Bouveret, E., Rigal, A., Lazdunski, C., and Bénédetti, H. (1997) Mol. Microbiol. 23, 909-920[CrossRef][Medline] [Order article via Infotrieve]
  23. Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508[Medline] [Order article via Infotrieve]
  24. Chanal, A., Santini, C.-L., and Wu, L.-F. (1998) Mol. Microbiol. 30, 674-676[CrossRef][Medline] [Order article via Infotrieve]
  25. Settles, A. M., Yonetani, A., Baron, A., Bush, D. R., Cline, K., and Martienssen, R. (1997) Science 278, 1467-1470[Abstract/Free Full Text]
  26. Weiner, J. H., Bilous, P. T., Shaw, G. M., Lubitz, S. P., Frost, L., Thomas, G. H., Cole, J., and Turner, R. J. (1998) Cell 93, 93-101[Medline] [Order article via Infotrieve]
  27. Rachubinski, R. A., and Subramani, S. (1995) Cell 83, 525-528[Medline] [Order article via Infotrieve]
  28. Budisa, N., Steipe, B., Demange, P., Eckerskorn, C., Kellermann, J., and Huber, R. (1995) Eur. J. Biochem. 230, 788-796[Abstract]
  29. Wolfe, P. B., Rice, M., and Wickner, W. (1985) J. Biol. Chem. 260, 1836-1841[Abstract]
  30. Baba, T., Jacq, A., Brickman, E., Beckwith, J., Taura, T., Ueguchi, C., Akiyama, Y., and Ito, K. (1990) J. Bacteriol. 172, 7005-7010[Medline] [Order article via Infotrieve]
  31. Jacobi, A., Rossmann, R., and Böck, A. (1992) Arch. Microbiol. 158, 444-451[Medline] [Order article via Infotrieve]
  32. Maneewannakul, S., Maneewannakul, K., and Ippen-Ihler, K. (1994) Plasmid 31, 300-307[CrossRef][Medline] [Order article via Infotrieve]


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