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The Haloferax volcanii FtsY Homolog Is Critical for Haloarchaeal Growth but Does Not Require the A Domain

Alex Haddad, R. Wesley Rose, and Mechthild Pohlschröder^*

Department of Biology, University of Pennsylvania, 201 Leidy Laboratories, 415 South University Ave., Philadelphia, Pennsylvania 19104

Received 30 November 2004/ Accepted 11 March 2005

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
The targeting of many Sec substrates to the membrane-associated translocation pore requires the cytoplasmic signal recognition particle (SRP). In Eukarya and Bacteria it has been shown that membrane docking of the SRP-substrate complex occurs via the universally conserved SRP receptor (Sr/ß and FtsY, respectively). While much has been learned about the archaeal SRP in recent years, few studies have examined archaeal Sr/FtsY homologs. In the present study the FtsY homolog of Haloferax volcanii was characterized in its native host. Disruption of the sole chromosomal copy of ftsY in H. volcanii was possible only under conditions where either the full-length haloarchaeal FtsY or an amino-terminally truncated version of this protein lacking the A domain, was expressed in trans. Subcellular fractionation analysis of H. volcanii ftsY deletion strains expressing either one of the complementing proteins revealed that in addition to a cytoplasmic pool, both proteins cofractionate with the haloarchaeal cytoplasmic membrane. Moreover, membrane localization of the universally conserved SRP subunit SRP54, the key binding partner of FtsY, was detected in both H. volcanii strains. These analyses suggest that the H. volcanii FtsY homolog plays a crucial role but does not require its A domain for haloarchaeal growth.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

 
All living organisms can be classified into three domains: the Eukarya, the Bacteria, and the Archaea (45). While the archaea are morphologically similar to the bacteria, at the molecular level they exhibit a combination of unique as well as bacterial and eukaryotic characteristics (18, 31). Recent whole-genome analyses of numerous archaea have expanded our understanding of this domain and provided insight into phylogenetic relationships, putative metabolic pathways, and cellular processes (3, 14, 37, 38). Furthermore, a more complete understanding of the Archaea has proven to have important implications for our understanding of cellular functions conserved in Eukarya and Bacteria (21, 34). This has been demonstrated in studies examining archaeal protein translocation via the universally conserved Sec pathway, which is thought to be responsible for the translocation of the majority of proteins into and across the endoplasmic reticular (ER) or cytoplasmic membranes in eukaryotes and prokaryotes, respectively (19, 35). Specifically, the notable achievement of the crystal structure of an archaeal Sec translocation pore, the first such structure obtained from any organism, has greatly contributed to the understanding of this universally conserved pore in all organisms (42). However, in addition to homologs of the universally conserved Sec components, archaea possess a mixture of homologs of bacterial and eukaryotic translocation components (29, 31). This observation, as well as the fact that homologs of translocation ATPases from Bacteria or Eukarya are lacking (e.g., SecA and Kar2p/BiP, respectively), suggest unique features of the archaeal protein translocation pathway (29, 31). Studying the significance of the similarities and differences among the Sec pathways of Archaea, Bacteria, and Eukarya will further our understanding of translocation in all the domains.

Recent in vivo and in vitro analyses of the protein translocation mechanisms of Haloferax volcanii, a halophilic archaeon amenable to genetic and biochemical analyses, have complemented genomic and structural analyses of the archaea (19, 35). The composition of the archaeal SRP is more similar to that of eukaryotes than the composition of bacterial SRP (10, 29, 35). In contrast, the composition of the eukaryotic SRP receptor (SR), a component of the ER membrane that docks the SRP nascent chain to the membrane, shows less similarity to its archaeal homolog (29). The eukaryotic SR is a heterodimer composed of Sr and a membrane protein, Srß, that anchors the Sr subunit to the ER membrane. In contrast, bacteria and archaea seem only to contain a homolog of the Sr subunit, called FtsY (29, 32).

Despite the lack of a prokaryotic Srß homolog, FtsY is found in both cytoplasmic and membrane-associated pools in the bacterial and archaeal species examined to date (6, 20, 25). FtsY is a member of the universally conserved SRP subfamily of GTPases whose members are characterized by the presence of a four alpha-helical bundle (termed the N domain) and immediately followed by the catalytic GTPase region (termed the G domain) (8). Both of these domains are involved in recognition of the universally conserved protein component of the SRP, SRP54 (termed Ffh in bacteria), and play critical roles in SRP-dependent protein translocation (9, 12). Many, though not all, examples of FtsY include an additional amino-terminal domain, named the "A" domain (9, 11, 12, 15). This name originated from the largely acidic character of the first example of FtsY to be described, that from Escherichia coli. Although not universal, many FtsY examples also have acidic A domains, including FtsY homologs from the haloarchaea (A. Haddad and M. Pohlschröder, unpublished observations).

In the absence of obvious membrane spanning regions in FtsY, it had been thought that the amino-terminal A domain mediated the interaction of FtsY with the membrane (7, 23, 24). However, this view recently has been challenged by several observations. First, sequence alignments reveal that in contrast to the strongly conserved N and G domains, the A domain is poorly conserved (29). Moreover, several bacterial and archaeal FtsY homologs lack an A domain (9, 11, 12). Second, the essential role of the A domain has been reevaluated in light of recent observations in E. coli, demonstrating that expression of FtsY lacking an A domain functionally complements disruption of the full-length protein (11). The amino-terminally truncated construct also associated with the E. coli cytoplasmic membrane, albeit less efficiently than full-length FtsY (11). Finally, in vitro analysis of purified proteins interacting with H. volcanii inverted vesicles showed that both the full-length haloarchaeal FtsY homolog and a version of FtsY lacking the A domain were able to associate with these membrane preparations (19). Interestingly, additional in vitro experiments suggested that the A domain may be involved in recruiting the SRP to the haloarchaeal cytoplasmic membrane (19).

We wished to further characterize the archaeal FtsY homolog in vivo. In the present study, we have employed a recently developed gene knockout strategy to demonstrate in vivo that the H. volcanii FtsY homolog is crucial for growth (2). Furthermore, we show that a haloarchaeal FtsY construct lacking the amino-terminal A domain is able to complement a chromosomal ftsY deletion and that in both strains the SRP is targeted to the membrane. Thus, although the archaeal SRP-dependent pathway shares similarities with that of both eukaryotes and bacteria, the archaeal FtsY appears in vivo to be more similar to the E. coli FtsY.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents. Reagents, including antibiotics, were purchased from Sigma-Aldrich (St. Louis, MO) except where noted. 5-Fluoroorotic acid (5-FOA) was from Toronto Research Chemicals (North York, Canada). The H. volcanii cosmid library was generously provided by R. L. Charlebois (Department of Biology, University of Ottawa) (5). Anti- dihydrofolate reductase (DHFR) antibodies were a kind gift from M. Mevarech (Tel Aviv University, Tel Aviv, Israel). Antibodies against the H. volcanii FtsY homolog were generously provided by J. Eichler (Ben Gurion University of the Negev, Beersheva, Israel) (19). Antibodies recognizing the H. volcanii SRP54 homolog were described previously (35). Protease inhibitors (pepstatin and leupeptin) were from Roche Applied Science (Indianapolis, IN). The enhanced chemiluminescence (ECL+) detection system was from Amersham Pharmacia (Piscataway, NJ). All enzymes were from New England Biolabs (Beverley, MA) unless otherwise indicated. Anti-penta-His antibodies and DNA purification systems (QIAquick and QIAprep) were from QIAGEN (Valencia, CA). RQ1 DNase I was from Promega (Madison, WI). NuPage gel system and reagents were from Invitrogen (Carlsbad, CA). Blotting membranes (polyvinylidene difluoride [PVDF]-plus) were from GE Osmonics Inc. (Minnetonka, MN).

Strains and growth conditions. Archaeal strains used in this study are listed in Table 1. Unless otherwise indicted, H. volcanii strains were routinely grown with aeration at 45°C in rich medium (RM) supplemented with novobiocin (0.3 µg/ml) or 5-FOA (150 µg/ml) when required (22). For gene integration, Casamino acid medium (CA) was used and supplemented with novobiocin as above, when needed (2). E. coli strains MP12 or XL1-Blue (Stratagene, La Jolla, CA), were routinely grown at 37°C in NZCYM supplemented with ampicillin (100 µg/ml) as required (30, 36).

Construction of ftsY deletion strains. The ftsY ORF in H. volcanii was deleted using a recently developed counter-selection scheme (2). Briefly, a targeting construct is created using genomic regions flanking the ORF to be targeted that are cloned in tandem into a nonreplicative plasmid upstream of the pyrE2 gene cassette. The targeting construct is subsequently transformed into H. volcanii strain WR480, a uracil auxotroph; thus, selection in the absence of uracil will generate a strain in which the targeting plasmid is integrated within either the upstream or downstream genomic regions flanking the targeted ORF (i.e., cointegrant or "pop-in"). The cointegrant strain is then grown without selection for a limited number of generations, allowing for recombination to take place, and subsequently plated on 5-FOA to select against the presence of the pyrE2 gene cassette. Under these conditions, recombination eliminating the pyrE2 gene occurs in one of two ways: (i) it removes the targeted gene, or (ii) it resolves the original plasmid. PCR was used to generate 860-bp DNA fragments that contained 800 bp of flanking genomic DNA (either upstream or downstream) along with 60 bp of the ftsY ORF. These were amplified from H. volcanii chromosomal DNA using primers designed to amplify the upstream (ftsYUP860-For and ftsYUP860-Rev) and downstream (ftsYDN860-For and ftsYDN860-Rev) flanking regions. The PCR product representing the upstream fragment was first digested with EcoRI and KpnI, agarose gel-purified (QIASPIN) and ligated to plasmid DNA (pGB70) that had been digested with EcoRI and KpnI and treated with calf intestinal phosphatase (CIP). Resulting plasmids, harboring the upstream ftsY fragment were digested with KpnI, CIP treated, and subsequently ligated to the PCR product representing the ftsY downstream fragment that had been digested with KpnI and gel purified. The resulting plasmid containing the ftsY downstream flanking sequence in the desired orientation, here named pAH-k1, was first passed through a dam mutant E. coli host (DL739) prior to transforming H. volcanii strain WR480, as previously described (17). Transformants were plated on CA medium to select for cointegrant strains (e.g., strain AH-co1). Independent colonies were then grown in liquid RM for 48 h at 40°C to allow for recombination at the ftsY locus and subsequently plated on RM supplemented with 5-FOA. Genotypes of isolated clones were determined by colony PCR using primers flanking the targeted region on the chromosome (ftsYscreen-For and ftsYscreen-Rev).

Expression of FtsY · 6xHis derivatives in H. volcanii. The ftsY ORF was modified by introducing a nonapeptide sequence encoding a six-histidine epitope tag (pro-pro-gly-his₆) in frame at the carboxy terminus using PCR amplification from the H. volcanii cosmid library (ftsYWT-For and ftsY · 6xHis-Rev). The amplification product was first purified from the reaction mixture (QIASPIN), digested with NcoI and KpnI, gel purified, and finally ligated to pNP15 plasmid DNA (Table 1), which had previously been digested with NcoI and KpnI, treated with CIP, and gel purified (pAH-x1). This created plasmid pAH-x1. Amino-terminal truncations of the H. volcanii FtsY homolog were designed removing either the predicted A domain [amino acids 2 to 164, to create FtsY(NG) · 6xHis], or both the A and N domains [amino acids 2 to 238, to create FtsY(G) · 6xHis]. These constructs were created identically to the full-length construct to generate plasmids pAH-x2 and pAH-x3, respectively, except that different forward primers were used in the initial PCR [for FtsY(NG) · 6xHis, the ftsYNG-For primer was used and for FtsY(G) · 6xHis, the ftsYG-For primer was used]. Each plasmid was passed through a dam mutant E. coli host, transformed into the H. volcanii cointegrant strain AH-co1, and subsequently plated on CA medium supplemented with novobiocin. In this way, both the ftsY targeting vector (pAH-k1), already integrated at the chromosomal ftsY locus, and the plasmid designed to supply the ftsY · 6xHis variants in trans were maintained. Independent colonies from strains harboring each plasmid were then grown in liquid RM containing novobiocin for 48 h to allow for recombination at the ftsY locus and subsequently plated on medium containing novobiocin and 5-FOA. 5-FOA-resistant strains were genotyped using PCR as described above. To confirm the expression of the plasmid encoded FtsY constructs, whole-cell lysates were prepared from isolated transformants by growth in liquid culture (10 ml) with novobiocin to an OD₆₀₀ of between 0.6 and 0.8. One ml of cells were pelleted at 4,500 x g and lysed directly in 90 µl to 120 µl of NuPage lithium dodecyl sulfate (LDS) sample buffer supplemented with reductant, according to the manufacturer's instructions, and incubated for one hour at 37°C prior to polyacrylamide gel electrophoresis (PAGE).

H. volcanii translation inhibition. H. volcanii strain AHwt20 was treated with the translational inhibitor puromycin sulfate as described by Ring et al. with minor modifications (33). Briefly, 50 µl of a late log phase AHwt20 culture grown in RM supplemented with novobiocin was transferred to 10 ml of Hv-min medium, supplemented with uracil (50 µg/ml), novobiocin (0.3 µg/ml), and methionine assay medium (250 µg/ml [Difco 242310]) (1). Once grown to late log phase the culture was diluted 1:100 in Hv-min with supplements and grown to mid-log phase, at which time it was divided into two cultures, which were incubated for an additional 20 min, one in the absence the other in the presence of 70 µl/ml of puromycin sulfate. Cytoplasmic and membrane fractions were then prepared from a 10-ml aliquot of each culture and analyzed by sodium dodecyl sulfate (SDS)-PAGE and Western blot analysis. The efficacy of the puromycin treatment was confirmed via metabolic labeling similar to the method described previously (33). Briefly, aliquots of the cultures, following incubation in the presence or absence of inhibitor, were labeled with [³⁵S]methionine (>1,000 Ci/mmol; NEG-709A; Office of Radiation Safety, University of Pennsylvania) for 5 min, precipitated with 10% (vol/vol) trichloroacetic acid (TCA), and then subjected to SDS-PAGE and visualized via phosphoimaging (Molecular Dynamics).

Subcellular fractionation of H. volcanii. H. volcanii strains grown in RM supplemented with novobiocin were harvested at mid- to late-log phase (OD₆₀₀ of 0.5 to 0.8) at 4,500 x g at 4°C for 10 min. Cells (5 ml) were washed once in basal salt medium (BSM; 3.5 M NaCl, 15 mM MgSO₄ · 7H₂O, 50 mM KCl, and 50 mM Tris [pH 7.2]), and cell pellets were resuspended in 0.5 ml BSM containing protease inhibitors (pepstatin, 1 µg/ml, and leupeptin, 2 µg/ml). Cells were lysed via sonication on ice at setting "50" using a Dynatech Sonic Dismembrator Model 150 equipped with a microtip probe using eight pulses of 3 s in 5-s intervals. Cellular debris was removed via two successive spins at top speed in a microcentrifuge at 4°C. The supernatant was then spun at 85,000 rpm for 30 min at 4°C (Beckman TLA 100.2) to separate the cytoplasmic and membrane fractions. The cytoplasmic fraction was then precipitated with 10% (vol/vol) trichloroacetic acid (TCA) for 30 min on ice. Precipitated protein was collected at top speed (20,800 x g) in a microcentrifuge at 4°C for 30 min, washed with cold 80% (vol/vol) acetone, and centrifuged again as before. Following removal of the acetone, the pellets were allowed to air dry briefly prior to resuspension in protein sample buffer. The membrane pellet was washed once with cold BSM containing protease inhibitors and subsequently resuspended in 75 µl of double-distilled H₂0 containing protease inhibitors. Both membrane and cytoplasmic fractions were solubilized in NuPage LDS sample buffer for one hour at 37°C prior to PAGE. For analysis of the H. volcanii SRP54 homolog, SDS sample buffer was used and electrophoresis was otherwise identical.

Western blot analysis. Proteins were resolved using the bis-Tris NuPage gel electrophoresis systems (Invitrogen). Proteins were transferred to Magna nylon membranes using a Bio-Rad trans-blot SD semidry transfer cell for 30 min at 15 V, according to the manufacture's instructions. Primary antibody (anti-FtsY, 1:2,000; anti-penta His, 1:2,000; anti-SRP54, 1:1,000; and anti-DHFR1, 1:7,000) and secondary antibody incubations (horseradish peroxidase conjugated [HRP] sheep anti-mouse IgG, 1:8,000, or donkey anti-rabbit immunoglobulin G [IgG] [both from Amersham Biosciences], 1:10,000) were performed in 3% (wt/vol) bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 0.1% (vol/vol) Tween-20 for one hour at room temperature. Washes were with PBS containing 0.1% (vol/vol) Tween-20. Membranes were processed with the ECL Plus Western Blotting detection system (Amersham Biosciences) according to the manufacturer's instructions. Immunoreactive bands were visualized by exposure to Super Rx Fuji Medical X-ray film (Fuji Photo Film, Co., Ltd., Tokyo, Japan). For quantification purposes, developed films were scanned and individual bands were quantified using the public domain software Image J (1.30v; Wayne Rasband, National Institutes of Health; http://rsb.info.nih.gov/ij). Band intensities were determined using integration of the area underlying each band peak.

Nucleotide sequence accession number. The nucleotide sequence for the ORF encoding the H. volcanii ftsY homolog has been deposited in GenBank under accession number AY187867.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
The H. volcanii ftsY homolog is critical for growth. Our in vivo characterization of the H. volcanii FtsY homolog began with the disruption of the ftsY locus using a counterselection strategy recently developed for this organism (2). Similar to approaches developed for use in Saccharomyces cerevisiae, this scheme takes advantage of the ability to positively or negatively select for the activity of orotate phosphoribosyl transferase (ORPTase, a component of the uracil biosynthetic pathway) by either requiring growth in the absence of uracil (for positive selection) or growth in the presence of the toxic compound 5-FOA (for counterselection). Briefly, in a pyrE2 strain (WR480, Table 1) a nonreplicative plasmid containing pyrE2 (the gene encoding the major H. volcanii ORPTase) was first targeted to the ftsY locus via homologous recombination with cloned ftsY flanking sequences. Plasmid cointegrants were selected by uracil prototrophy. Resolution of the cointegrated plasmid was selected in a subsequent step by growth on media containing both uracil and 5-FOA. This second recombination event can either regenerate the intact gene or generate a knockout at the chromosomal locus.

As confirmed by PCR, we were able to generate H. volcanii cointegrant strains that had integrated the targeting plasmid adjacent to the ftsY locus (AH-co1, Table 1). However, PCR analysis of 27 5-FOA-resistant colonies arising from two independent counterselections of AH-co1 revealed that all clones had retained ftsY intact (Fig. 1A and data not shown). These results strongly suggest that FtsY is crucial for H. volcanii growth under these conditions. It is possible, however, that the retention of chromosomal ftsY in these strains occurred because the integrated plasmid was unable to recombine in a manner that would allow for its deletion. To eliminate this possibility, AH-co1 was transformed with a plasmid designed to express full-length FtsY that has a six-histidine epitope tag at the carboxy terminus (FtsY · 6xHis, pAH-x1, Table 1 and Fig. 1B) creating strain AH-co1x1. When strain AH-co1x1 was exposed to 5-FOA, recombinants containing either the intact or disrupted ftsY gene were recovered [Fig. 1C, (AHwt20) and (AHwt21), respectively]. A total of 29 clones were screened with 6 retaining intact ftsY loci (data not shown). This result confirms that the haloarchaeal FtsY homolog is important for H. volcanii growth and, moreover, that ftsY · 6xHis encodes a functional protein.

View larger version (47K): [in this window] [in a new window]   FIG. 1. Disruption of the H. volcanii chromosomal ftsY locus requires expression of either FtsY or FtsYNG in trans. (A) Schematic diagram of the H. volcanii ftsY chromosomal locus targeted for disruption. The boxed region represents the H. volcanii ftsY locus (1,371 bp) targeted for deletion using flanking genomic information (emboldened lines). Arrows flanking this area of recombination denote positions of oligonucleotide primers used to determine the genotype of the resulting strains. (B) Representations of the epitope-tagged derivatives that were expressed in trans during gene targeting. Letters within the boxes designate the respective domains. The small white boxes at the carboxy terminus of each construct (following the G domain) represent the location of the 6x His tag. (C) Amplicons generated using the PCR with primers denoted in (A) from H. volcanii strains as indicated ( denotes ftsY deletion; wT denotes wild-type ftsY locus). (D) Western blot analysis of H. volcanii whole-cell lysates prepared from strains analyzed in (C), using an antibody recognizing the H. volcanii FtsY homolog.

To ascertain the importance of the H. volcanii FtsY A domain for its function, we repeated the complementation strategy similar to the one described above, testing the functionality of the tagged full-length FtsY by transforming the cointegrant strain AH-co1 with a plasmid expressing ftsYNG · 6xHis (Fig. 1B and Table 1, pAH-x2). Using PCR screening, genotypic analysis of 5-FOA-resistant colonies revealed strains possessing either a disrupted (AHng09) or an intact (AHng10) ftsY locus (Fig. 1C; 2 out of 24 clones screened contained disrupted ftsY loci). These results strongly suggested that ftsYNG · 6xHis provided in trans could functionally complement a chromosomal ftsY deletion. To confirm the absence of wild-type FtsY in strain AHng09, immunoblot analysis of whole-cell lysates was performed, using an antibody that recognizes the H. volcanii FtsY NG domain (Fig. 1D). Western blots clearly showed the lack of a specific band detecting the full-length FtsY in strain AHng09, suggesting that the endogenous ftsY was not expressed in this strain. Furthermore, it identified a specific immunoreactive band at 32 kDa, the predicted molecular mass of FtsYNG · 6xHis in strain AHng09. Additionally, comparison of the growth rates of AHwt20 and AHng09 under several different conditions (in both rich and minimal media at 45°C and 30°C [the lower temperature is known to reveal a temperature-sensitive phenotype linked with certain secretion defects]) revealed no differences between these strains (28) (data not shown).

Finally, we asked whether a gene that encodes a version of FtsY that lacks both the A and the N domains (ftsYG · 6xHis) from the same expression vector that was used for the previous assays (pAH-x3, Table 1 and Fig. 1B) could complement a chromosomal ftsY deletion. Genotypic analysis of 5-FOA-resistant colonies derived from these experiments (a total of 24 colonies were analyzed) indicated that, although the protein was properly expressed, the ftsY locus in each one of these strains remained intact (Fig. 1 C and D). These results not only confirm that the highly conserved N domain is required for the H. volcanii FtsY function but also demonstrate that disruption of the chromosomal ftsY locus in H. volcanii, complemented by ftsYNG · 6xHis, was not due to secondary effects introduced by the expression vector.

Membrane-associated FtsY is detected in the presence and absence of the A domain. Both in vivo and in vitro analyses suggest that in most of the prokaryotic systems examined to date, FtsY is present in the cytosol as well as associated with the membrane (6, 19, 25). Consistent with these results, immunoblot analysis of H. volcanii membrane and cytoplasmic fractions, using an -FtsY antibody, detected this protein in both fractions in wild-type strain WFD11 (Fig. 2A).

View larger version (32K): [in this window] [in a new window]   FIG. 2. The H. volcanii FtsY A domain is dispensable for its membrane association. Western blot analysis of cytoplasmic and membrane fractions prepared from a nontransformed H. volcanii strain (WFD11) and ftsY strains expressing different FtsY constructs. Standardized equivalents of cytoplasmic (c) and membrane (m) fractions were analyzed using (A) anti-H. volcanii FtsY antibodies or (B) antibodies recognizing the H. volcanii cytoplasmic protein DHFR1.

ftsY

Cellular localization of SRP in H. volcanii wild-type and ftsY strains. (i) The H. volcanii SRP54 homolog can be detected in both the haloarchaeal cytoplasm and membrane. Cellular localization studies suggest that subpopulations of SRP54, the universally conserved SRP component that interacts with the SR, is associated with the ER membrane (43). Consistent with these results, a subpopulation of the Acidianus ambivalens SRP54 homolog and the Halobacterium halobium 7SRNA homolog have been shown to be associated with the archaeal cytoplasmic membranes (16, 25). However, while in vitro membrane binding assays also point to a membrane-associated pool of the H. volcanii SRP54 homolog (19), previous results from H. volcanii cellular fractionations only identified this SRP subunit in the cytoplasm (41). Because of the widely accepted role of FtsY in facilitating membrane association of the SRP complex, we first reexamined the localization of the H. volcanii SRP54 homolog in the AHwt20 strain. Western blot analysis of cytoplasmic and membrane fractions revealed the presence of an immunoreactive band migrating at 52 kDa in both fractions (Fig. 3A). To determine whether the immunoreactive bands were specific to the H. volcanii SRP54 homolog, an H. volcanii strain expressing a his-tagged SRP54 (strain WR6c) was analyzed by Western blot (35). Analysis of cytoplasmic and membrane fractions from this strain revealed identical immunoreactive bands using both anti-penta-his and anti-SRP54 antibodies (data not shown). These results confirm that the bands are specific to the H. volcanii SRP54 homolog and demonstrate that, similar to organisms of other domains, a pool of the archaeal SRP54 is stably associated with the cytoplasmic membrane.

View larger version (37K): [in this window] [in a new window]   FIG. 3. The H. volcanii SRP54 does not require the FtsY A domain for its membrane association. Cytoplasmic (c) and membrane (m) fractions prepared from the AHwt20 and AHng09 ftsY strains were subjected to Western blot analysis using (A) an antibody recognizing the H. volcanii SRP54 homolog or (B) antibodies recognizing the H. volcanii cytoplasmic protein DHFR1.

H. volcanii ftsY

View larger version (18K): [in this window] [in a new window]   FIG. 4. Sequence conservation and divergence of FtsY homologs. (A) Boxed representation of FtsY homolog domains. Shading of boxes represents increasing conservation from light to dark. Blackened areas labeled G1 to G4 represent highly conserved motifs in the G domain involved in nucleotide binding and catalysis (9, 12). The N domain is defined as a four alpha-helical bundle whose presence is universally conserved in the SRP family of GTPases (8). The A domain is the least conserved portion of these proteins, with little to no conservation in primary sequence among those representatives that possess such a domain (11, 29). (B) Amino acid alignments of portions of the Thermus aquaticus and E. coli FtsY homologs are adapted from that of Egea et al. (9). The amino terminus of the H. volcanii FtsYNG · 6xHis construct used in this study was aligned to these sequences based on the strongly conserved "ALLEADV" motif present within the N domain (the underlined portion of amino acid sequences in boldface type) (9, 12). The arrow above the H. volcanii sequence denotes the first amino acid position of the A domain truncation used in in vitro analyses (19). The sequence boundary for the E. coli protein was extended from amino acid 201 in Egea et al. (9) to amino acid position 196 to include those amino acids in the E. coli protein that were recently shown necessary to complement a deletion of the chromosomal ftsY in this organism (11).

View larger version (31K): [in this window] [in a new window]   FIG. 5. Inhibition of protein synthesis results in decreased membrane-associated SRP54 without a change in membrane-associated FtsY. Western blot analysis of membrane (m) and cytoplasmic (c) fractions prepared from H. volcanii strain AHwt20 grown in the absence (–) or presence (+) of puromycin for 20 min prior to fractionation. Complete cessation of protein synthesis under these conditions were confirmed using metabolic protein labeling (see Materials and Methods and data not shown). Western blots are shown using antibodies recognizing H. volcanii SRP54 (A) or FtsY (B).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

ftsY

Interestingly, we observed that in addition to the wild-type ftsY, a gene encoding an H. volcanii FtsY construct lacking the N-terminal A domain (ftsYNG · 6xHis) could complement the chromosomal ftsY knockout. In contrast, a ftsY construct expressing only the G domain of this protein could not complement an ftsY deletion. Taken together, these results demonstrate that both the N and G domains of the H. volcanii FtsY homolog are essential for its function, while the A domain is dispensable under the growth conditions tested in this study. The fact that a truncated FtsY consisting of only the G domain was insufficient for complementation is not surprising given the universal conservation of both the N and G domains in all GTPases of the 5RP family. The current view of the organization of these two domains is that they act as a single functional unit (44).

While we were able to demonstrate that expression of FtsYNG in trans was sufficient to complement the loss of endogenous ftsY, the relative number of ftsY deletion recombinants and wild-type recombinants following counterselection was lower than that obtained when expressing the wild-type gene in trans. It is possible that the H. volcanii ftsY knockout strain, complemented by the expression of ftsYNG · 6xHis (AHng09), is less fit than the wild-type strain and is therefore recovered less frequently. However, comparative analysis of the H. volcanii ftsY knockout strain complemented with full-length ftsY · 6xHis (AHwt20) to that complemented with ftsYNG · 6xHis (AHng09) suggest that the lack of the A domain does not severely affect its function. First, the growth rates between the two strains were the same at either 45°C or 30°C (data not shown); second, both FtsY · 6xHis and FtsYNG · 6xHis proteins associated with the H. volcanii cytoplasmic membrane; and finally, the H. volcanii homolog of SRP54, the critical binding partner of FtsY, was found to be associated with the cytoplasmic membrane in both the AHwt20 and AHng09 strains. While the plasmid-encoded H. volcanii FtsY variants were expressed under the control of a heterologous promoter, Western blot analysis using an antibody against the H. volcanii FtsY homolog suggest that the FtsY constructs and the chromosomally encoded FtsY are produced at comparable levels (Fig. 1D, compare endogenous FtsY seen in lanes 4 and 5 to the plasmid encoded protein seen in lane 1). Thus, it is unlikely that expression of any of the FtsY variants from their native promoter would significantly change these results.

Our data are in agreement with in vitro analyses of an H. volcanii FtsYNG construct, which demonstrated that the haloarchaeal FtsY A domain was dispensable for its association with H. volcanii-inverted membrane vesicles (19). However, these studies also suggested that, unlike the full-length FtsY, the H. volcanii FtsYNG construct was unable to promote association of the H. volcanii SRP54 homolog to the vesicles (19). Conversely, our data suggest that a subpopulation of SRP54 is associated with the membrane. It should be noted, however, that the construct used in those studies lacked a portion of the N domain immediately adjacent to the highly conserved "ALLEADV" motif (Fig. 4), a motif that is thought to contribute to SRP54/FtsY binding (9, 12). In contrast, the truncation of the complementing FtsYNG construct characterized here, while lacking the A domain, includes 27 amino acids amino terminal to this motif (see Fig. 4B).

Our finding that the H. volcanii FtsY homolog is required for cellular growth but that the A domain is dispensable supports recent analyses of an amino-terminally truncated E. coli FtsY construct (11). These findings resurrect two important questions about this protein. First, "What is the function of the FtsY A domain?" As shown above, deleting this poorly conserved domain does not seem to be detrimental in either bacteria or archaea. In fact, several archael and bacterial orthologs seem to lack this domain, such as Pyrobaculum aerophilum strain IM2 and Thermotoga volcanium, respectively (9, 11, 12) (Haddad and Pohlschröder, unpublished). It had been long thought that the A domain was important for promoting association of FtsY with the cytoplasmic membrane (7, 23, 24). However, membrane binding of both the E. coli and H. volcanii FtsYNG constructs and complementation of ftsY via expression of ftsY · 6xHis clearly refute earlier proposals suggesting its involvement in membrane association. These results raise a second question: "How do FtsY homologs that do not possess obvious membrane anchors associate with the cytoplasmic membrane?" Unlike in the eukaryotes, where the FtsY homolog SR interacts with the membrane protein Srß, an Srß homolog or other membrane proteins with which FtsY interact have not been identified (29). Furthermore, translation inhibition resulted in the release of a subpopulation of the membrane-bound SRP54 but not of FtsY from the H. volcanii cytoplasmic membrane. This result suggests that FtsY membrane association is not solely due to its association with translating SRP-ribosome nascent chain complexes. Finally, we have also performed copurification studies of H. volcanii membrane preparations utilizing the 6x His tag appended to each of the expressed FtsY variants in order to detect specific interacting components. Although the use of 6x His-tagged proteins has been successful in our laboratory in the copurification of the SRP components (35), we have not identified any additional proteins that stably interact with FtsY · 6xHis (Haddad and Pohlschröder, unpublished). This is consistent with in vitro studies in E. coli suggesting that the primary interaction of FtsY with cytoplasmic membranes occurs with phospholipids, as well as genetic screens that have also not revealed interacting membrane protein components (7, 24, 39, 40). Interestingly, the ambiguity of a membrane anchor for the SR is not restricted to prokaryotes. While Sr forms a heterodimer with the transmembrane protein Srß, truncation of the yeast Srß transmembrane anchor does not affect growth in the yeast Saccharomyces cerevisiae (26). Furthermore, in vitro protein translocation using purified components is unaffected when the transmembrane anchor within Srß is removed (13). Thus, identifying mechanisms of bacterial and archaeal FtsY membrane association might also lead to a better understanding of the significance of the membrane localization of the eukaryotic SRP receptor.

	ACKNOWLEDGMENTS

Support was provided to R.W.R. by a predoctoral fellowship from the American Heart Association (reference no. 0110093U) and to M.P. by a National Science Foundation grant (reference no. MCB-0239215).

	FOOTNOTES

 
* Corresponding author. Mailing address: (215) 573-2283. Fax: (215) 898-8780. E-mail: pohlschr{at}sas.upenn.edu.

Present address: Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111.

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