Co-translocation of a Periplasmic Enzyme Complex by a Hitchhiker
Mechanism through the Bacterial Tat Pathway*
Agnès
Rodrigue
,
Angélique
Chanal
,
Konstanze
Beck§¶,
Matthias
Müller§, and
Long-Fei
Wu
From the
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 |
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 |
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 |
Strains, Plasmids, and Growth Conditions--
Bacterial strains
and plasmids used in this study are listed in Table
I. To construct the
hybOABC
mutant, the 3421-base pair XhoI-BglII fragment
containing hybOABC was replaced by the promoterless cassette
encoding
-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.
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 (
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 (
hybOABC/hyb(A-G)+) and
ENF1/pHyb411 (
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 |
Cloning of the hyb Operon and Construction of the
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
(
hya,
hybBC, and
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 ( hybOABC;
panel A) carrying plasmids pHyb14
(hyb(O-G)+), pHyb411
(hybO+), or pHyb41
(hyb(O-B,D-G)+) or from NH1
( hybOABC- 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.
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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
nik-
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).
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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 (
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.
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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.
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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 ( hybOABC/hybO+; lane
1), ENF1/pHyb55
( hybOABC/hyb(pelBssO-G)+;
lane 2), and ENF1/pHyb14
( 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
hybOABC (lanes 3 and 4),
secYts- hybOABC (lanes 1, 2, 5, and
6), and secAts- 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 hybOABC (lanes 1, 4, and
7), secYts- hybOABC (lanes 2, 5, and
8), and secAts- 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.
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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 |
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,
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.
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.
 |
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