From the Department of Life Sciences, Ben Gurion University of the Negev, Beersheva 84105, Israel
Received for publication, October 22, 2002, and in revised form, January 30, 2003
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ABSTRACT |
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Although protein secretion occurs
post-translationally in bacteria and is mainly a cotranslational event
in Eukarya, the relationship between the translation and translocation
of secreted proteins in Archaea is not known. To address this question,
the signal peptide-encoding region of the surface layer glycoprotein
gene from the Haloarchaea Haloferax volcanii was fused
either to the cellulose-binding domain of the Clostridium
thermocellum cellulosome or to the cytoplasmic enzyme
dihydrofolate reductase from H. volcanii. Signal
peptide-cleaved mature versions of both the cellulose-binding domain
and dihydrofolate reductase could be detected in the growth medium of
transformed H. volcanii cells. Immunoblot analysis
revealed, however, the presence of full-length signal peptide-bearing
forms of both proteins inside the cytoplasm of the transformed cells. Proteinase accessibility assays confirmed that the presence of cell-associated signal peptide-bearing proteins was not due to medium
contamination. Moreover, the pulse-radiolabeled signal peptide
cellulose-binding domain chimera could be chased from the cytoplasm
into the growth medium even following treatment with anisomycin,
an antibiotic inhibitor of haloarchaeal protein translation. Thus,
these results provide evidence that, in Archaea, at least some secreted
proteins are first synthesized inside the cell and only then
translocated across the plasma membrane into the medium.
Correct protein localization is essential for the proper working
of the cell. In the case of proteins destined to reside beyond the
cytoplasm, such localization requires transfer across lipid-based membranes. In both bacteria and Eukarya, secreted proteins are synthesized as precursors, or "preproteins," containing a cleavable N-terminal extension referred to as the signal peptide
(SP)1 (1). The SP, not found
in the mature polypeptide, serves to target the preprotein to
membrane-embedded translocation complexes (2). Although the signal
peptides of bacterial and eukaryal secretory proteins, incorporating a
positively charged region followed by a hydrophobic core and
then a polar cleavage region, are often highly similar and even
interchangeable (3), the temporal relationship between secretory
protein translation and translocation differs between these two domains
of life. In bacteria, secreted proteins are translocated
post-translationally, i.e. once most, if not all, of the
protein has been translated in the cytoplasm (4, 5). In contrast, the
translocation of secretory proteins across the membrane of the eukaryal
endoplasmic reticulum, the topological homolog of the bacterial plasma
membrane, is coupled to protein translation (1), although
post-translational translocation has been reported in Eukarya
(6-8).
Although the process of protein translocation is well described in
bacteria and Eukarya (9-12), little is known about how proteins cross
the plasma membrane of Archaea, the third and most recently described
domain of life. Archaea encode components of the translocation
complexes found in the bacterial and eukaryal systems, and exported
archaeal proteins often contain signal peptides similar to those found
in the other two domains (13, 14). Numerous other aspects of the
archaeal secretion process have, however, yet to be addressed. In
particular, the interplay between translation and translocation during
archaeal protein export remains to be elucidated. Archaea contain a
signal recognition particle (SRP), the agent responsible for linking
translation and translocation in Eukarya (15), reminiscent of its
eukaryal counterpart (16). However, many Archaea also encode homologs
of SecDF (17), proteins that, in bacteria, serve to modulate the
membrane association of SecA, the translocation complex component
responsible for coupling ATP energy to the post-translational movement
of preproteins across the plasma membrane (18, 19). However, searches
of completed archaeal genomes have thus far failed to reveal an
archaeal SecA homolog. Thus, questions concerning the driving force of
archaeal translocation also remain unanswered.
Studies addressing the relationship between archaeal protein
translation and translocation to date have focused mainly on the
biosynthesis of the Halobacterium salinarum
multispanning membrane protein bacterioopsin. Based on the
co-sedimentation of 7 S RNA and bacterioopsin mRNA with
membrane-bound polysomes, as well as more recent in vivo
kinetic analysis, a cotranslational SRP-dependent mode of
protein translocation is suggested in Archaea (20, 21). In contrast,
studies following the membrane insertion of a chimeric version of
bacterioopsin expressed in Haloferax volcanii reported that
the fusion protein is first synthesized in the cytoplasm and only then
inserted into the membrane (22). Studies relying on newly synthesized
bacterioopsin as a reporter of the relation between translation and
translocation in Archaea must be viewed, however, with caution, given
this protein's unusually short 13-residue signal peptide that lacks a
hydrophobic core and contains negatively charged glutamate residues,
rather than the positively charged amino acids found in most signal
peptides (20), as well as the possibility of a dedicated system for
bacterioopsin membrane insertion (22). Moreover, the interaction
between translation and translocation of a membrane protein may
differ in the case of protein secretion.
Thus, to elucidate the temporal relation between translation and
translocation during archaeal protein secretion, this work addressed
the heterologous expression of two chimeric preproteins composed of the
SP of the surface layer glycoprotein (SLG) of H. volcanii
fused either to the cellulose-binding domain (CBD) of the
Clostridium thermocellum cellulosome or to the cytoplasmic H. volcanii enzyme dihydrofolate reductase-1 (DHFR-1). The
molecular composition of the 34-amino acid-long SP of the SLG is
similar to those of secretory preproteins in other domains of life (2, 3) and, as previously observed, includes a cleavage site recognized by
type I signal peptidases (25). Our studies reveal that, in H. volcanii, these chimeric preproteins are first completely
synthesized within the confines of the cell and are only then
translocated across the plasma membrane into the medium, with the SP
being most likely cleaved by the externally oriented,
membrane-bound signal peptidase.
Materials--
Ampicillin, anisomycin, cellulose, DNase I,
novobiocin, polyethylene glycol 600, and Triton X-100 were from Sigma.
Proteinase K was from Roche Molecular Biochemicals (Mannheim, Germany).
Yeast extract was from Pronadisa (Madrid, Spain), and Tryptone was from U. S. Biochemical Corp. Molecular mass markers and horseradish peroxidase-conjugated goat anti-rabbit antibodies were from Bio-Rad. Redivue 35S radiolabeling mixture (>1000 Ci/mmol) and an
ECL kit were from Amersham Biosciences (Buckingham, UK).
Organisms and Growth Conditions--
The H. volcanii
methionine/cysteine auxotrophic strain WR341 (22) and H. volcanii WR441, lacking the hdrA gene encoding DHFR-1
(24), were grown aerobically at 40 °C (22). H. volcanii SX/CBD bearing the pWL-CBD plasmid encoding the CBD moiety of the
C. thermocellum cellulosome (25) was grown in the same
medium, to which novobiocin (1 µg/ml) was added. All strains were
obtained from Moshe Mevarech (Tel Aviv University) and are
characterized in Table I.
Plasmid Construction and Transformation--
Recombinant
plasmids for expression of chimeric proteins bearing the SP of the
H. volcanii SLG fused either to the CBD from C. thermocellum or to DHFR-1 from H. volcanii were
constructed as follows (see Fig. 1A). The first 34 codons of
the gene encoding the H. volcanii SLG were PCR-amplified
from plasmid pUC18-SLG (obtained from Manfred Sumper, University of
Regensberg) (23) using the forward primer 5'-CTTGTCATGACAAAGCTCAAA-3'
and the reverse primer 5'-ATATCCATGGTCGCGGCGGCA-3', designed to
introduce BspHI and NcoI restriction sites at the
5'- and 3'-ends of the fragment, respectively. The PCR-amplified
product was subcloned at the NcoI site of plasmid pWL-CBD
containing the cbd gene encoding the C. thermocellum cellulosome CBD fused to the haloarchaeal PrR16
promotor. This cloning yielded plasmid pWL-SP-CBD containing a fusion
of the 3'-end of the SLG signal peptide-encoding sequence ahead of the
cbd gene, separated by a threonine-encoding codon linker, and behind the PrR16 promotor. The SP-encoding PCR-amplified fragment was also subcloned at the NcoI site of plasmid pHE1-DHFR-1
containing the hdrA gene (encoding H. volcanii
DHFR-1) fused to the PrR16 promotor, yielding the pHE1-SP-DHFR-1
plasmid. H. volcanii cells were transformed with pWL-SP-CBD
and pHE1-SP-DHFR-1 essentially as described (26) and selected in
H. volcanii medium supplemented with novobiocin (1 µg/ml).
The pWL-SP-CBD plasmid was used to transform H. volcanii
WR341 cells to create strain SX/SP-CBD, whereas pHE1-SP-DHFR-1 was used
to transform H. volcanii WR441 to create strain
WR441/SP-DHFR-1. Stable propagation of the plasmids was verified by
plasmid re-isolation and characterization by PCR analysis and DNA sequencing.
Radiolabeling--
H. volcanii cells transformed with
plasmid pWL-SP-CBD (30 ml) were grown to A550 = 0.6, harvested (8000 × g, 15 min), resuspended in 30 ml of minimal medium (27), and grown aerobically at 40 °C for
12 h. For pulse radiolabeling, [35S]Met (143 µCi)
was added to 10 ml of the cell culture, and 1-ml aliquots were removed
at various intervals and immediately centrifuged (8000 × g, 10 min, 4 °C). The supernatant (containing the medium) was removed and kept on ice. The cells in the pellet were lysed with 1 ml of lysis buffer (1% Triton X-100, 1.8 M NaCl, and 50 mM Tris-HCl, pH 7.2). For pulse-chase radiolabeling,
[35S]Met (143 µCi) was added to 10-ml cultures in
minimal medium for 3-5 min, after which time unlabeled methionine was
added to a final concentration of 1 mM. In some cases,
anisomycin (20 µg/ml) was added during the final minute of
radiolabeling. Aliquots were removed immediately prior to and at
various intervals following the addition of the unlabeled methionine
and processed as described above. In both labeling protocols, DNase I
(3 µg/ml) was then added to the fractions. The mixtures were rocked
(10 min, room temperature), after which time 50 µl of 10% (w/v)
cellulose were added. After 60 min of rocking at room temperature, the
suspension was centrifuged (1000 × g, 3 min); the
supernatant was discarded; and the cellulose pellet was washed with 2 M NaCl and 50 mM Tris-HCl, pH 7.2. This washing
procedure was repeated twice. After the final wash, the cellulose beads
were centrifuged (2200 × g, 3 min); the supernatant
was removed; and the cellulose pellet was resuspended in 40 µl of
SDS-PAGE sample buffer. The samples were then boiled for 5 min and
centrifuged (2200 × g, 5 min) to release any
cellulose-bound proteins. The proteins from cytoplasmic and media
samples captured and later released from the cellulose beads were
examined by 15% SDS-PAGE and viewed by fluorography using Kodak X-Omat
film or a Fuji phosphorimager.
Other Methods--
Cells were separated from the growth medium
by centrifugation (8000 × g, 15 min). The supernatant
(containing the medium and secreted proteins) was removed. The pelleted
cells were resuspended in fresh growth medium and again collected by
centrifugation. This washing step was repeated twice. In some cases,
the separated cells were treated with proteinase K (1 mg/ml final
concentration, 30 min, 40 °C), in the presence or absence of 1%
Triton X-100. Subcellular fractionation was achieved by osmotic lysis
of cells upon transfer into 1 ml of water, addition of DNase I (3 µg/ml), and separation of the soluble and membrane fractions by
ultracentrifugation (Sorvall Discovery M120 ultracentrifuge, S120ATS
rotor, 73,000 rpm, 10 min, 4 °C). Immunoblotting was performed using
antibodies raised against the C. thermocellum CBD (obtained
from Arie Admon, Technion Israel Technology Institute), against
H. volcanii DHFR-1 (obtained from Moshe Mevarech), or
against the H. volcanii SLG (28). Antibody binding was
detected using horseradish peroxidase-conjugated goat anti-rabbit
antibodies and enhanced chemiluminescence. Protein concentration was
determined using Bradford reagent (Bio-Rad) with bovine serum albumin
as the standard. Densitometry was performed using IPLab Gel software
(Signal Analytics Corp., Vienna, VA). For amino acid sequencing,
proteins were electrotransferred to polyvinylidene fluoride membranes
and subjected to analysis at the Protein Sequencing Facility of the
Weizmann Institute of Science (Rehovot, Israel).
SP-bearing Fusion Proteins Are Detected Inside Transformed H. volcanii Cells--
To assess the relation between protein translation
and translocation in Archaea, H. volcanii WR341 cells were
transformed with plasmid pWL-SP-CBD, encoding a chimeric protein
composed of the SP of the H. volcanii SLG fused to the
cbd gene, encoding the C. thermocellum
cellulosome CBD (Fig. 1B). The
synthesis and secretion of the chimeric reporter preprotein SP-CBD
(22.5 kDa) were assessed by separating the transformed cells from their
growth medium and analyzing the total protein content of each fraction by SDS-PAGE. At the same time, cellular fractions and growth media of
the background WR341 and CBD-expressing cells were similarly analyzed.
Coomassie staining revealed the presence of a protein corresponding in
size to CBD (18.5 kDa) only in the growth medium of the
SP-CBD-transformed strain (Fig.
2A, lane 3). To
confirm the identity of the secreted protein as the processed form of the SP-CBD fusion, i.e. CBD, N-terminal sequencing was
performed. Such analysis revealed the first 6 amino acids as TMANTP,
corresponding to the first 5 amino acids of CBD preceded by the
additional threonine residue introduced during the cloning of plasmid
pWL-SP-CBD (see "Experimental Procedures"). Examination of the
protein profiles of the cellular fractions of the three strains failed
to reveal any visible differences (Fig. 2A, lanes
4-6), indicating that the presence of the mature CBD in the
medium of SP-CBD-expressing cells was not the result of medium
contamination by ruptured cells in which accumulation or overexpression
of the SP-CBD precursor had occurred. Thus, the results show that the
SP-CBD-transformed strain successfully expresses and secretes the
SP-CBD preprotein. At some point in the translocation event, the SP
likely undergoes signal peptidase-mediated cleavage.
To confirm that the CBD was secreted only from those cells transformed
to express the SP-CBD chimera, background and CBD- and
SP-CBD-expressing cells and their growth media were separated and
subjected to immunoblotting using antibodies raised against the CBD. As
observed upon Coomassie staining (Fig. 2A), the antibodies detected only CBD protein in the growth medium of the strain
transformed to express the SP-CBD fusion protein (Fig. 2B,
lane 3). Immunoblotting of the cellular fractions of the
three cultures also revealed the presence of the 18.5-kDa CBD in the
CBD-expressing cells (Fig. 2B, lane 5); no such
band was stained in the nontransformed cells (lane 4).
Surprisingly, however, three bands were also recognized by the
antibodies in the cellular fraction of the SP-CBD-expressing cells
(Fig. 2B, lane 6). The uppermost of these bands
corresponds in size to the SP-fused version of the CBD (black
arrow). This suggests that SP-CBD is first completely synthesized
inside the cells and only then secreted across the plasma membrane into
the growth medium. The two additional smaller bands found inside the SP-CBD-transformed cells apparently represent partial breakdown products of the preprotein, likely resulting from proteolytic cleavage
of the preprotein inside the cell, in agreement with earlier studies
showing proteolysis of heterologously expressed fusion proteins in
H. volcanii (22, 29, 30). Subcellular fractionation of the
SP-CBD-expressing cells revealed that the preprotein and its derived
breakdown products were restricted to the cytoplasmic fraction (data
not shown).
To verify that the presence of the SP-bearing precursor in transformed
H. volcanii cells was not related to the use of a
non-haloarchaeal protein entity such as the CBD, H. volcanii
cells were transformed with plasmid pHE1-SP-DHFR-1, encoding a chimera
of the H. volcanii SLG signal peptide fused to H. volcanii DHFR-1, one of the two versions of the cytoplasmic enzyme
in this species (24). Expression of the chimeric preprotein in the
transformed cells was verified by immunoblotting of background, WR441,
and WR441/SP-DHFR-1 strains using anti-DHFR-1 antiserum. The antibodies
recognized only a protein band corresponding in size to DHFR-1 (17.8 kDa) in the growth medium of the WR441/SP-DHFR-1 culture (Fig.
2C, lane 3). This indicates that H. volcanii cells are capable of secreting a normally cytoplasmic
protein to which the SP of the SLG has been attached. In addition, the
antibodies also recognized a slightly smaller band, likely a
degradation product of DHFR-1. Next, the cellular fraction of each
culture was probed with the anti-DHFR-1 antibodies. As expected, DHFR-1
was stained by the antibodies in the control WR341 cells (Fig.
2C, lane 4), yet was not detected in H. volcanii WR441, a DHFR-1 deletion strain (lane 5).
Examination of the cellular fraction of the SP-DHFR-1-transformed cells
revealed the presence of the SP-bearing DHFR-1 preprotein (21.8 kDa),
as well as a few smaller bands (Fig. 2C, lane 6).
Moreover, as is the case with SP-CBD-transformed cells, the presence of
cellularly associated, smaller anti-DHFR-1 antibody-labeled bands
indicates that proteolytic cleavage of the preprotein also occurs
inside the cell. Given the higher degree of SP-DHFR-1 breakdown
compared with SP-CBD degradation, reduced amounts of secreted mature
DHFR-1 would be expected. Accordingly, Coomassie staining failed to
reveal secreted DHFR-1 in the growth medium of WR441/SP-DHFR-1 cells (data not shown).
Cell-associated SP-CBD and SP-DHFR-1 Are Not Externally
Accessible--
Experiments were next undertaken to confirm that the
existence of cell-associated SP-bearing CBD and DHFR-1 preproteins was not due to small amounts of contaminating growth medium containing unprocessed secreted forms of the chimeric proteins being captured along with the isolated cells. In these studies, isolated
SP-CBD-expressing cells were challenged with proteinase K to digest any
SP-bearing CBD possibly present in the medium that may have been
captured along with the isolated cells. As reflected in Fig.
3 (upper panel), the protease
treatment failed to affect SP-CBD levels, as detected by antibody
staining (compare lanes 1 and 2). If, however,
cell integrity was disrupted by pretreatment with 1% Triton X-100, thereby allowing access of the protease to the cell's interior, the
SP-CBD precursor was completely digested (Fig. 3, lane 3). The level of the SP-CBD-derived degradation product was relatively unaffected by the protease treatment, most likely because of a protease-resistant conformation assumed by this polypeptide (31). Protease treatment also failed to affect the levels of cell-associated SP-DHFR-1 or its breakdown products, as detected by antibody staining (Fig. 3, middle panel, compare lanes 1 and
2). Inclusion of 1% Triton X-100 in the reaction led,
however, to a complete digestion of both the SP-DHFR precursor and its
breakdown products (Fig. 3, middle panel, lane
3). Finally, in control experiments, the ability of proteinase K
to digest externally oriented H. volcanii cell-surface
markers such as the SLG was confirmed both in WR441/SP-DHFR-1 cells
(Fig. 3, lower panel) and in WR341/SP-CBD cells (data not shown).
Full-length Nascent SP-CBD Is First Synthesized Inside the Cell and
Only Then Secreted--
Although full-length SP-bearing precursor
proteins can be detected inside transformed cells, it is possible that
these polypeptides correspond to translocation-incompetent preproteins
that cannot be secreted and hence become entrapped within the cell. To
discount this possibility, a series of kinetic radiolabel-based studies were undertaken.
In pulse labeling experiments, SP-CBD-expressing cells were
metabolically 35S-radiolabeled. Samples were removed at
various intervals, and the labeled cells were separated from their
growth medium. Subsequently, the growth medium and solubilized cellular
fractions were incubated with cellulose. Cellulose interacts with the
CBD in a salt-independent manner (22), thereby allowing for
affinity-based precipitation of radiolabeled SP-CBD and of the secreted
mature CBD under the high salt conditions required for H. volcanii growth. As shown in Fig.
4A, analysis of
cellulose-captured proteins by SDS-PAGE and fluorography first revealed
the capture of the SP-CBD precursor in the cellular fraction close to
the onset of the radiolabeling period. SP-CBD-derived breakdown
products were also captured by cellulose in the cellular fractions. In
contrast, the processed mature form of the protein only appeared in the
medium following a delay of 3 min.
The delay between the appearance of the full-length SP-CBD precursor in
the cytoplasm and the detection of the processed CBD in the growth
medium could reflect the post-translational translocation of the
preprotein. Alternatively, it is possible that a low efficiency of
SP-CBD secretion would require that sufficient levels of CBD accumulate
in the growth medium before detection of the secreted protein would be
possible. In this latter scenario, translocation need not occur
subsequent to translation, but rather could occur concomitantly. To
discount this possibility, SP-CBD-transformed cells were subjected to
pulse-chase radiolabeling. Here, cells were metabolically radiolabeled
with [35S]methionine for 3 min, followed by the addition
of a large excess of unlabeled methionine to initiate the chase phase
of the reaction. Aliquots were removed at various intervals and
processed as described above. Examination of the cellular fractions
revealed that the level of SP-CBD preprotein substantially decreased
following the onset of chase (Fig. 4, B and C).
Over the same period, an increase in the level of mature CBD in the
growth medium was measured. Thus, the pulse-chase radiolabeling studies
support the view that post-translational translocation of the
SP-bearing CBD occurs in the transformed H. volcanii cells.
The cellular fraction also contained a substantial amount of
SP-CBD-derived breakdown products, in agreement with earlier studies
addressing the expression of fusion proteins by H. volcanii
(29, 32).
To further confirm that secretion of SP-CBD is temporally distinct from
the translation of the protein, an additional experiment was performed
in which pulse-chase radiolabeling was repeated; however, now, 20 µg/ml anisomycin was added during the final minute of the pulse phase
of the experiment. Anisomycin is an effective inhibitor of protein
synthesis in H. volcanii (14). Indeed, preliminary studies
revealed that, at the anisomycin concentration employed in these
studies, protein synthesis in the SP-CBD-transformed cells was no
longer detectable even as soon as 30 s following exposure to the
antibiotic (Fig. 5A). However,
treatment with the antibiotic had no effect on SP-CBD secretion
because, as reflected in Fig. 5B, the radiolabeled mature
CBD could be readily detected in the growth medium of the
anisomycin-treated preprotein-expressing cells. Moreover, anisomycin
treatment did not prevent the rapid drop in cellular SP-CBD levels.
Quantitation of mature CBD levels in the growth medium from three
separate experiments confirmed that protein secretion continued well
after the arrest of translation by the antibiotic (Fig. 5C).
Therefore, these results confirm that SP-CBD is first translated inside
the cell and only then translocated across the plasma membrane into the
growth medium, with SP cleavage occurring along the way.
In Archaea, a variety of proteins must be translocated into and
across the plasma membrane, such as membrane proteins, secreted enzymes, and the components of the protein-based surface layer. Presently, numerous aspects of the archaeal protein export process remain to be elucidated, including whether archaeal translocation occurs post-translationally, as in bacteria, or cotranslationally, as
in Eukarya. To investigate this question, H. volcanii cells were transformed to express chimeric preproteins formed from the SP of
the H. volcanii SLG fused either to the CBD of the C. thermocellum cellulosome, thus allowing for salt-insensitive
cellulose-based purification of the preprotein and its secreted
product, or to H. volcanii DHFR-1 to monitor the secretion
of a haloarchaeal cytoplasmic protein now designed for export. The
transformed cells effectively expressed and secreted the chimeric
preproteins, as detected by the presence of SP-cleaved versions of the
CBD and DHFR-1 in the respective growth media. However, examination of the protein content of the isolated cells revealed the presence of the
full-length preproteins, suggesting that secretion of both the foreign
CBD and native DHFR-1 had occurred post-translationally. Kinetic
radiolabeling experiments confirmed that secretion indeed took place
only after the chimeric preproteins had been translated in the cytosol
of transformed H. volcanii cells. Furthermore, arrest of
continued protein translation had no effect on the secretion of
previously pulse-radiolabeled SP-CBD.
An alternative conclusion that might be drawn from these experiments is
that the observed post-translational secretion reflects the cell's
efforts to cope with massive overexpression of the plasmid-encoded
fusion proteins. This scenario is, however, unlikely due to several
reasons. The plasmid employed for transformation of H. volcanii cells includes the non-inducible constitutive PrR16 promotor (29, 32). Accordingly, no differences were noted in the growth
rates or cell yields of the background and recombinant strains, effects
that would be expected upon massive protein overproduction. Indeed,
Coomassie staining of the cellular protein contents in Fig.
2A failed to reveal strongly overexpressed proteins
migrating at the positions of the CBD or SP-CBD in the transformed
H. volcanii cells compared with the nontransformed
background strain. Moreover, densitometric comparison of the
intensities of anti-DHFR-1 antibody-reactive bands in background and
SP-DHFR-1-transformed strains (Fig. 2C) revealed that the
transformed cells contained only 5-fold more immunoreactive material
than the background cells. This value is likely an overestimate, given
that it also includes the contribution of SP-DHFR-1 breakdown products
that accumulated inside the cell and would not be secretion-competent.
Thus, like earlier studies relying on this vector that failed to report
large-scale overexpression of encoded proteins (22, 29), it appears
that massive overexpression of the fusion proteins is not the case in
the present study. As such, it is unlikely that massively overexpressed
SP-CBD and SP-DHFR-1 saturate a normally employed cotranslational
secretion pathway and are thus secreted following translation. Indeed,
regardless of the level of expression of the fusion proteins, the
observation that protein secretion occurs at similar levels in the
presence or absence of an inhibitor of haloarchaeal protein synthesis
offers strong support that the post-translational secretion observed in
these studies reflects a normal and constitutive cellular process.
The SP of the H. volcanii SLG employed to target the CBD and
DHFR-1 for export in this study displays a molecular composition similar to that borne by SPs of Sec-dependent preproteins
in Eukarya and bacteria (3). Given the similarity of SP composition, it is reasonable to predict that the archaeal preproteins in this study
also rely on the Sec machinery for their secretion. In Eukarya, the
vast majority of secretory proteins are cotranslationally targeted to
Sec translocation sites in the endoplasmic reticulum membrane,
i.e. the translocon, via the SRP pathway (15). Despite the
fact that the archaeal SRP is, in many ways, similar to its eukaryal
counterpart (16), it seems that archaeal protein secretion has more in
common with bacteria, the other prokaryal domain, as both bacteria and
Archaea apparently rely on a post-translational mode of protein secretion.
In the bacterial Sec pathway, SecB or other chaperones
post-translationally deliver secretory protein precursors to SecA, peripherally bound to the membrane at translocation sites based on the
trimeric SecYEG complex (11, 33). Exploiting its ATP-hydrolyzing activity, SecA then drives the transfer of its preprotein cargo across
the plasma membrane. Although Archaea encode versions of SecYEG (13,
14, 34) (and possibly SecB (13)), searches of completed archaeal
genomes have yet to detect an archaeal SecA homolog. The existence of a
structural homolog of SecA in Archaea, not detectable by current
bioinformatic approaches, cannot, however, be ruled out. Therefore, it
is noteworthy that despite the apparent absence of SecA in Archaea,
several archaeal species encode homologs of SecDF (17), components
shown to modulate the membrane association of SecA in Escherichia
coli (18, 19).
Examples of post-translational SecA-independent translocation via the
Sec pathway do exist. In yeast, post-translational protein export
relies on various cytoplasmic chaperones for the delivery of
preproteins to the translocon (35). Once delivered, the endoplasmic reticulum resident protein BiP employs ATP energy to pull the preprotein into the endoplasmic reticulum lumen (36, 37). It is
unlikely that Archaea would rely on a similar mechanism, given the
improbability of nucleotide being present in sufficient quantities on
the exterior surface of the plasma membrane, the topological homolog of
the luminal face of the endoplasmic reticulum membrane. Moreover, many
of the chaperones involved in eukaryal post-translational translocation
are absent in several archaeal species (38). As such, components
involved in the post-translational targeting and translocation of
secretory proteins across the archaeal plasma membrane may remain to be
identified. With this in mind, it is noteworthy that two recent studies
have reported extensive use of the alternative Tat secretion pathway,
involved in post-translational translocation in bacteria, in
Haloarchaea (39, 40).
The successful ability of the fused H. volcanii SLG signal
peptide to target both the CBD and DHFR-1 to the growth medium may
reveal other general aspects of the translocation process in this
species and possibly in other Archaea. The H. volcanii SLG,
the sole component of the proteinaceous shell surrounding the cell,
spans the plasma membrane via a single transmembrane domain (23). Thus,
the finding that the same SP can direct both a membrane-anchored
protein as well as secreted proteins to the cell exterior suggests that
both classes of proteins rely on the same translocation apparatus.
Finally, although only a very limited number of cases have been
addressed, it is tempting to speculate that, in Archaea, protein secretion occurs post-translationally, whereas membrane protein insertion occurs in a cotranslational SRP-dependent manner.
However, not all archaeal membrane proteins may rely on the SRP pathway for their insertion, as possibly exemplified by studies addressing the
homologous (20, 41) and heterologous (22) expression of bacterioopsin.
Indeed, the SRP pathway is implicated in the membrane insertion of only
a subset of bacterial plasma membrane proteins (42). Given the
availability of inverted membrane vesicles from H. volcanii
(43), reconstituted H. volcanii SRP (44), and purified
SP-CBD2 and
SP-bacterioopsin-DHFR-1 (45) preproteins, the stage is now set to
reconstitute archaeal protein translocation in vitro and to
address these questions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Strains and plasmids used in this study
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, schematic representation of plasmids
pWL-SP-CBD and pHE1-SP-DHFR. See "Experimental Procedures" for a
description of the plasmids and their construction. B,
schematic representation of SP-CBD and SP-DHFR-1. The amino acid
sequences of the SP of the SLG and the first 10 amino acids of the CBD
and DHFR-1 moieties are shown. The position of the signal peptidase
cleavage site is marked, as is the position of the addition threonine
residue introduced during construction of the plasmids. In addition,
the basic (n), hydrophobic (h), and cleavage
(c) regions of the SP are indicated.
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Fig. 2.
SP-bearing preproteins are detected in the
cytosol, whereas SP-cleaved proteins are found in the medium.
A, aliquots (1 ml) of cultures of H. volcanii
(WR341) and H. volcanii transformed to express either the
CBD or SP-CBD were separated into cellular and growth media fractions,
and each was examined by SDS-PAGE. Molecular mass markers (in
kilodaltons) are shown to the right of each panel. The black
arrow reflects the expected position of SP-CBD, whereas the
white arrow reflects the position of the CBD. B,
the separated cellular and media fractions of 200-µl aliquots of each
cell culture were probed with antibodies to CBD. The black
arrow reflects the position of SP-CBD, whereas the white
arrow reflects the position of the CBD. The positions of molecular
mass markers (in kilodaltons) are shown on the right. C,
aliquots (200 µl) of cultures of H. volcanii (WR341),
H. volcanii lacking the gene encoding DHFR-1 (WR441), and
WR441 transformed to express DHFR-1 (WR441/SP-DHFR-1) were separated
into cellular and growth media fractions, and each was electroblotted
and probed with antibodies to DHFR-1. The black arrow
reflects the position of SP-DHFR-1, whereas the white arrow
reflects the position of DHFR-1.
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Fig. 3.
SP-bearing preproteins are protected from
added protease. H. volcanii cells transformed to
express SP-CBD (upper panel) or SP-DHFR-1 (middle
and lower panels) were exposed to the presence of proteinase
K in the absence (lane 2) or presence (lane 3) of
1% Triton X-100. In lane 1, neither protease nor detergent
was added. The samples were then subjected to immunoblotting with
antibodies against the CBD (upper panel), DHFR-1
(middle panel), or the SLG (lower panel). The
positions of SP-CBD, SP-DHFR-1, and the SLG are denoted in the
upper, middle, and lower panels,
respectively.
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Fig. 4.
SP-CBD is first translated in the cytosol and
then secreted into the growth medium. H. volcanii cells
transformed to express SP-CBD were metabolically
35S-pulse-radiolabeled or radiolabeled in a pulse-chase
paradigm. Aliquots were removed at various intervals and transferred to
ice-cold tubes. Cellular and media fractions were separated, incubated
with cellulose, and examined by SDS-PAGE and fluorography.
A, shown are the results from pulse radiolabeling.
B, shown are the results from pulse-chase radiolabeling. In
both A and B, the position of SP-CBD is denoted
by the black arrow, whereas the position of the CBD is
denoted by the white arrow. C, the fluorograph of
the pulse-chase radiolabeling was densitometrically analyzed to
determine the intensity of the SP-CBD ( ) and CBD (
) bands.
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Fig. 5.
Arrest of continued protein translation does
not interfere with SP-CBD secretion. A, H. volcanii cells were metabolically 35S-radiolabeled (15 µCi/ml, 5 min) before or after pretreatment with anisomycin (20 µg/ml) for varying lengths of time. At each time point, the samples
were trichloroacetic acid-precipitated and examined by SDS-PAGE and
fluorography. The fluorograph was purposely overexposed to confirm the
inhibition of protein translation by the antibiotic. The positions of
molecular mass markers (250, 150, 100, 75, 50, 37, 25, 15, and 10 kDa)
are shown on the right. B, anisomycin (20 µg/ml) was added
to SP-CBD-transformed H. volcanii cells during the final
minute of a 5-min 35S radiolabeling pulse. Chase was then
initiated upon the addition of excess unlabeled methionine. Aliquots
were removed either immediately before or at various intervals
following the onset of the chase. The cells and growth medium were
isolated from each time point, incubated with cellulose, and examined
by SDS-PAGE and fluorography. C, shown is the densitometric
quantitation of the level of CBD secreted into the growth medium. The
values, expressed as -fold increase from the onset of chase (taken as
1.0), represent the means ± S.D. of two to three
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We acknowledge the technical assistance of Zvia Konrad.
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FOOTNOTES |
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* This work was supported by Israel Science Foundation Grant 291/99.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: Dept. of Life
Sciences, Ben Gurion University of the Negev, P. O. Box 653, Beersheva 84105, Israel. Tel.: 972-8646-1343; Fax: 972-8646-1710; E-mail: jeichler@bgumail.bgu.ac.il.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M210762200
2 V. Irihimovitch and J. Eichler, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: SP, signal peptide; SRP, signal recognition particle; SLG, surface layer glycoprotein; CBD, cellulose-binding domain; DHFR, dihydrofolate reductase.
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REFERENCES |
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