Post-translational Secretion of Fusion Proteins in the Halophilic Archaea Haloferax volcanii*

Vered Irihimovitch and Jerry EichlerDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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.


                              
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Table I
Strains and plasmids used in this study

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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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).


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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.

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.


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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 (open circle ) bands.

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.


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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

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.

    ACKNOWLEDGEMENT

We acknowledge the technical assistance of Zvia Konrad.

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

The abbreviations used are: SP, signal peptide; SRP, signal recognition particle; SLG, surface layer glycoprotein; CBD, cellulose-binding domain; DHFR, dihydrofolate reductase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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