Correspondence to Per O. Ljungdahl: plju{at}licr.ki.se
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Introduction |
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A highly conserved heterotrimeric complex of membrane proteins, i.e., the Sec61 complex in eukaryotes and SecY complex in bacteria forms the protein-conducting channel. The crystal structure of the Methanococcus jannaschii SecY translocon has recently been determined (van den Berg et al., 2004). In contrast to previous models of the translocon, the protein-conducting channel appears to be exclusively contained within SecY, the largest subunit of the heterotrimeric complex and the Sec61p homologue. According to this new information, the protein-conducting channel, although perhaps quite flexible, is too small to accommodate multiple TMS. Despite unresolved questions regarding the structure of an active Sec61/SecY translocon in vivo (Alder and Johnson, 2004; Rapoport et al., 2004), these new structural findings highlight persistent questions regarding how hydrophobic TMS of partially integrated polytopic proteins avoid inappropriate inter- and intramolecular interactions before the complete translocation and insertion of remaining TMS. Such inopportune interactions would likely hamper proper folding, and it has been suggested that membrane-localized chaperones exist to prevent such disadvantageous interactions (Lecomte et al., 2003; Alder and Johnson, 2004; Rapoport et al., 2004). In bacteria, the membrane protein YidC appears to play a chaperone-like role in the folding of lactose permease (LacY; Nagamori et al., 2004). To date, no such proteins have been identified in eukaryotes.
We use yeast amino acid permeases (AAPs) as models to address how polytopic membrane proteins insert into the ER membrane, fold, and subsequently enter ER-derived transport vesicles. The AAPs of Saccharomyces cerevisiae make up a conserved family of 18 proteins with 12 TMS (Gilstring and Ljungdahl, 2000). AAPs belong to the secondary transporter amino acid/polyamine/organocation protein family, members of which are found in bacteria, archaea, fungi, plants, and animals (Saier, 2000). All but one of the yeast AAPs function at the PM using the H+ gradient to drive the translocation of amino acids into the cell. One member of the AAP family with a large NH2-terminal extension, Ssy1p, functions as a core component of the SPS-sensor of extracellular amino acids (Forsberg and Ljungdahl, 2001).
AAPs are initially inserted into the ER membrane after which they are cotransported together with other secreted proteins from the ER to the Golgi via COPII-coated transport vesicles (Kuehn et al., 1996). The integral ER membrane component Shr3p is an accessory protein (Herrmann et al., 1999; Lee et al., 2004; Nyman et al., 2004) that is required for AAPs to exit the ER (Ljungdahl et al., 1992). The secretory block observed in cells carrying mutations in SHR3 is specific, other membrane-bound and secretory cargo exit the ER and are targeted to their correct intracellular locations (Ljungdahl et al., 1992; Kuehn et al., 1996). Shr3p itself does not exit the ER (Kuehn et al., 1996). Shr3p is a well-conserved protein in fungi, and homologues function similarly and interchangeably in Schizosaccharomyces pombe (Martínez and Ljungdahl, 2000) and Candida albicans (Martínez and Ljungdahl, 2004).
Shr3p is not required for insertion of AAPs; each of the TMS of the archetypal general AAP (Gap1p) integrates into the ER membrane in the correct orientation independently of Shr3p (Gilstring and Ljungdahl, 2000). The AAPs that accumulate in the membrane of the ER of shr3 null mutant cells do not induce the ER stress response pathway (Gilstring et al., 1999), indicating that the accumulated AAPs do not expose sequences that bind Kar2p, the homologue of mammalian BiP (Rutkowski and Kaufman, 2004). Despite having proper membrane topologies and not inducing ER stress, the AAPs in cells lacking Shr3p fail to be included in prebudding complexes and COPII transport vesicles (Kuehn et al., 1996, 1998).
Consistent with its specialized role in promoting the exit of AAPs from the ER, Shr3p physically associates with Gap1p, but not with other polytopic membrane proteins, such as Sec61p, Gal2p or Pma1p, in a transient complex that can be purified from detergent solubilized membranes (Gilstring et al., 1999). In addition, the COPII coatomer components Sec13p, Sec23p, Sec24p, and Sec31p copurify with the hydrophilic COOH-terminal domain of Shr3p (Gilstring et al., 1999). Based on the complete repertoire of interactions, it has been proposed that Shr3p functions to initiate the formation of transport vesicles in the vicinity of AAPs (Gilstring et al., 1999), perhaps by facilitating the presentation of ER exit sequences present in the COOH-terminal portion of AAPs (Malkus et al., 2002; Miller et al., 2003). Alternatively, it has been suggested that Shr3p acts as a "mismatch" chaperone (Levine et al., 2000). Mismatch chaperones are hypothetical entities that have been postulated to prevent inappropriate interactions between PM proteins during their residence in the ER, a membrane that is thought to be thinner than the average length of the hydrophobic TMS of PM proteins.
Here, we show that the primary function of Shr3p is to prevent the aggregation of AAPs thereby enabling them to fold correctly within the ER membrane. We also report that three additional ER components, Gsf2p, Pho86p, and Chs7p, act similarly to Shr3p, and their presence specifically prevents the aggregation of defined sets of polytopic membrane proteins, their cognate substrates. Strikingly, in cells lacking one of these ER proteins, we observe all-or-nothing effects, i.e., nearly quantitative aggregation and cross-linking that is limited to their cognate substrates. Our findings suggest that these ER proteins act as highly specialized membrane-localized chaperones that facilitate the folding of polytopic membrane proteins into their native tertiary membrane conformations, a requisite for ER exit.
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Results |
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Shr3p influences the propensity of AAPs to be cross-linked
To address the possibility that Shr3p functions as a membrane-localized chaperone we probed the physical status of AAPs in vivo by examining the susceptibility of Gap1p to form cross-links in the presence of two membrane-permeable cross-linking reagents, i.e., thiol-cleavable Dithiobis[succinimidyl propionate] (DSP) or UV-activated N-succinimidyl 6-[4'-azido 2'-nitrophenylamino]hexanoate (SANPAH; Fig. 3 A). In contrast to Gap1p in membranes prepared from SHR3 wild-type cells (Fig. 3 A, lanes 15), Gap1p in membranes from shr3 mutant cells formed extensive cross-links as evidenced by the almost complete absence of detectable monomeric forms of Gap1p (Fig. 3 A, lanes 610). The propensity of Gap1p to be cross-linked in shr3
mutant cells was specific (Fig. 3 B). Two ER resident proteins, dolichol phosphate mannose synthase (Dpm1p) and Sec61p, and the PM hexose transporter Hxt1p did not become cross-linked; each of these proteins consistently migrated as monomers independent of the presence or absence of Shr3p. The ability to efficiently cross-link AAPs only in the absence of Shr3p is consistent with Shr3p having a critical role in enabling AAPs to attain native conformations. It should be noted that whereas the amounts of AAP present as monomers is reproducible from experiment to experiment, the amounts of AAPs present as high molecular weight smears is variable. This is a consequence of well established problems associated with blotting high molecular complexes.
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Based on our novel understanding regarding the chaperone-like function of Shr3p, we examined the susceptibility of the cognate substrates of these accessory proteins to form cross-links. In contrast to Hxt1p in GSF2 wild-type cells (Fig. 7 A, lanes 15), Hxt1p in gsf2 mutant cells readily formed cross-links (Fig. 7 A, lanes 610). The propensity of Hxt1p to be cross-linked in gsf2
mutant cells was specific; Gap1p and Sec61p did not become cross-linked and migrated as monomers independent of the presence or absence of Gsf2p (Fig. 7 A, bottom two panels). Similarly, Pho84p exhibited Pho86p-dependent cross-linking (Fig. 7 B) and Chs3p exhibited Chs7p-dependent cross-linking (Fig. 7 C). Our data indicate that Gsf2p, Pho86p, and Chs7p function in a manner indistinguishable from Shr3p by acting to prevent inappropriate molecular interactions of their cognate substrates.
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Discussion |
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The observation that Gsf2p, Pho86p, and Chs7p function similarly to Shr3p (Fig. 7) suggests that polytopic membrane proteins require chaperone-like proteins to overcome common structural constraints associated with membrane insertion and folding. Strikingly, in cells lacking one of the four membrane-chaperones examined, cross-linking was found to be an almost all-or-nothing phenomenon. In wild-type cells, essentially none of the cognate substrates were cross-linked, whereas in isogenic null mutants lacking one of the membrane-chaperones, the propensity of cognate substrates to form cross-links was nearly complete. The quantitative nature of the requirement of Shr3p to prevent aggregation of AAPs was clearly observed using BN-PAGE. In SHR3 wild-type cells the AAPs were readily solubilized and migrated as monomers, whereas in shr3 null mutants the monomeric forms were barely detected (Figs. 4 and 5; Fig. 6 C). In no instance was dimers or trimers of AAPs observed in membranes isolated from wild-type cells, therefore in their native forms, these polytopic proteins are not likely to function as closely packed or covalently linked multimers.
Our data are reminiscent of recent results regarding the LacY in Escherichia coli (Nagamori et al., 2004). LacY depends on YidC, a member of a conserved family of membrane proteins (Abl3/Oxa1/YidC; Kuhn et al., 2003; Dalbey and Kuhn, 2004) to attain a native tertiary structure (Nagamori et al., 2004). Similar to Shr3p, YidC does not appear to be necessary for the insertion of the membrane spanning segments of LacY, but nonetheless, YidC must be present during translation of LacY for it to obtain a native conformation (Nagamori et al., 2004). Our findings of membrane-localized chaperones in yeast provide further evidence for the high degree of functional conservation of the process of membrane protein insertion in prokaryotic and eukaryotic cells.
Based on the all-or-nothing effect that we observe, it is likely that membrane-localized chaperones interact early with their cognate polytopic substrate proteins before the partitioning of all their TMS into the lipid phase of the ER membrane, and before the completely translated proteins are released from the Sec61 translocon. The crystal structures of bacterial PM transporters LacY and GlpT, each comprised of 12 TMS, have recently been elucidated (Abramson et al., 2003; Huang et al., 2003). The availability of these structures has clearly shown that interactions between TMS within the lipid phase of the membrane are not limited to immediately flanking TMS. Thus, it is possible that membrane-localized chaperones prevent TMS that do not normally interact in the mature protein from engaging in nonproductive interactions with flanking TMS as they sequentially partition into the membrane. Alternatively, the presence of membrane-localized chaperones may function to ensure the efficient insertion of discrete TMS into the membrane. It is known that exclusively hydrophobic TMS rapidly exit the protein-conducting channel of the translocon, and readily partition into the lipid phase of the membrane (Heinrich et al., 2000). In contrast, less hydrophobic TMS containing charged or polar residues partition into the membrane less readily, and are retained in close proximity to the translocon, as evidenced by their ability to cross-link to translocon associated proteins (Heinrich and Rapoport, 2003). Thus, in the case of AAPs, Shr3p may facilitate the partitioning of one or more of the TMS of AAPs that have charged residues (i.e., TMS IIVII; Gilstring and Ljungdahl, 2000). In the absence of Shr3p, the inability of these TMS to efficiently partition in the membrane could result in inappropriate interactions leading to aggregation.
The characterization of membrane-localized chaperones in yeast may provide a useful framework to better understand the expression of PM proteins controlling important biological processes in multicellular organisms. Several putative accessory proteins have been identified in cells of metazoan origin. For example, in mammalian cells, receptor-associated membrane proteins determine the intracellular transport and ligand specificity of G proteincoupled receptors (McLatchie et al., 1998; Bermak et al., 2001), and in Caenorhabditis elegans, a select subset of odorant receptors require the action of ODR-4 to be correctly localized to the PM (Dwyer et al., 1998; McClintock and Sammeta, 2003). In addition, the ER proteins BAP29/BAP31 have been postulated to affect the folding of a rather diverse set of membrane proteins and thereby influence their ability to exit the ER (Schamel et al., 2003). It is currently not known whether these proteins influence the membrane insertion and folding of their cognate substrates, however it would be interesting to apply the assays described here to examine the possibility that these metazoan accessory proteins function as membrane-localized chaperones.
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Materials and methods |
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DNA cloning
The sequences of oligonucleotide primers are available on request. The BamHI site in pPL210 (Ljungdahl et al., 1992) was destroyed by restriction and fill-in creating pJK5. Single-stranded pJK5 was used as a template to enable the introduction of novel BamHI sites into SHR3 by site-directed mutagenesis (Gilstring and Ljungdahl, 2000); oligonucleotides NT-BHI, L1-BHI, L2-BHI, L3-BHI, and CT-BHI were used to create plasmids pJK30pJK34 that carry BamHI sites inserted immediately after sequences corresponding to amino acids 1, 44, 87, 125, and 210, respectively. A BamHI-flanked SUC2 cassette from plasmid pFG112 (Gilstring and Ljungdahl, 2000) was individually inserted into each BamHI site creating plasmids pJK40pJK44, respectively. The sequences encoding the 50amino acid hydrophilic COOH-terminal domain of SHR3 were deleted using oligonucleotide CT and single-stranded pJK5 as a template. The resulting plasmid pJK36 encodes a truncated protein comprised of the first 160 aa of Shr3p (Shr3
CTp). Plasmid pJK60 was created by inserting a 4.3-kb SwaINotI fragment containing STP1
131 from pCA027 (Andréasson and Ljungdahl, 2002) into SwaINotI restricted pRS317. Plasmids pJK64 and pJK65 were created by inserting SalIXbaI fragments from pPL210 (SHR3) and pJK36 (shr3
CT) into SalIXbaI restricted pRS317, respectively.
Protein manipulations
The membrane topology of Shr3p was determined by assessing the glycosylation state of the topo-reporter cassette present in proteins expressed by plasmids pJK40 through pJK44 in strain FGY212 (shr3) (Gilstring and Ljungdahl, 2000). The intracellular location of Gap1p in JKY3 (sec12-1) and JKY4 (sec12-1 shr3
6) was determined by fractionation on 1260% sucrose gradients essentially as described previously (Egner et al., 1995). Cells were grown in SC at 22°C (permissive temperature) to an OD600 of 1.8, cells were harvested and washed twice in water. The washed cells were resuspended (OD600 of 1.8) in media containing allantoin (SAD) to induce Gap1p expression and incubated for 2 h at 34°C (nonpermissive temperature). Induced cells were collected by centrifugation, washed, and resuspended in 1 ml of lysis buffer (OD600 of 200). Cells were lysed by vigorous vortexing in the presence of glass beads (Gilstring et al., 1999). To remove unlysed cells and debris the lysates were centrifuged twice for 5 min at 2,000 g, and 1 ml of supernatant was layered onto gradients. A 50-µl aliquot of each fraction was mixed with equal amount of 3x SDS-PAGE sample buffer. Samples were heated for 10 min at 45°C, and proteins were resolved by SDS-PAGE and analyzed by immunoblotting.
Immunoblots were incubated as indicated with primary antibody in blocking buffer diluted as follows: rabbit -Shr3p, 1:1,000; rabbit
-Gap1p, 1:20,000; rabbit
-Agp1p, 1:10,000; rat
-HA monoclonal (Roche), 1:1500; mouse
-Dpm1p monoclonal (Molecular Probes), 4 µg/ml; rabbit
-Sec61p, 1:5,000; rabbit
-Hxt1p, 1:1,000; mouse
-MYC monoclonal, 1:1,000; mouse
-Pma1p monoclonal, 1:10,000; rabbit
-Kex2p, 1:1,000; and mouse
-GFP monoclonal, 1:1,000 (Roche). Immunoreactive bands were visualized by chemiluminescence emanating from HRP-conjugated to a secondary antibody;
rabbit Ig from donkey,
mouse Ig from sheep or
rat Ig from goat (Amersham Biosciences), using the LAS1000 system (Fuji Photo Film Co. Ltd.). The
-Gap1p and
-Agp1p antisera were provided by B. André. The
-Pma1p,
-Hxt1p,
-Kex2p, and
-Sec61p antibodies were obtained from J.P. Aris (University of Florida, Gainesville, FL), E. Boles (Goethe-Universitaet Frankfurt, Frankfurt, Germany), R. Fuller (University of Michigan Medical School, Ann Arbor, MI), and C. Stirling (University of Manchester, Manchester, UK), respectively.
Cross-linking
Cross-linking in the presence of either thiol cleavable DSP or UV-activated SANPAH cross-linkers (Pierce Chemical Co.) was performed as follows. Cells were grown to an OD600 of 0.81.0, harvested by centrifugation, washed twice in water, and resuspended at an OD600 of 150200 in PBS, pH 7.4, containing protease inhibitors (Boehringer). Cells were lysed by vortexing in the presence of glass beads, and unlysed cells and debris were removed by centrifugation. DSP cross-linking: 40 µl reactions containing 10 µg protein in PBS and the indicated concentration of DSP were incubated for 30 min at 22°C. Free reactive groups were quenched in the presence of 30 mM Tris-HCl buffer, pH 7.5, for 30 min at 22°C. Samples were treated with 40 mM DTT for 30 min at 37°C as indicated, untreated samples were kept on ice. SANPAH cross-linking: 30 µl reactions containing 8 µg protein in PBS and the indicated concentration of SANPAH were incubated for 30 min at 22°C in the dark. Reactions were quenched as above for 15 min at 22°C. Photoactivation was performed by exposing samples to 366 nm UV light for 10 min.
Blue nativePAGE
Cells were grown to an OD600 of 0.81.0, harvested by centrifugation, washed twice in water, and resuspended at an OD600 of 150200 in BN buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing protease inhibitors (Boehringer). Protein extracts were prepared as described for cross-linking. Proteins (1.8 mg ml1 in BN buffer) were solubilized with DM (Roche) at 4°C for 30 min, and a 0.1 vol of BN-PAGE sample buffer (5% Serva blue G, 100 mM BisTris, 500 mM 6-aminocaproic acid, and 80% glycerol, pH 7.0) was added. Proteins were separated on 515% BN gradient gels as described previously (Schägger et al., 1994). High molecular weight markers (Amersham Biosciences) were used as standards.
Microscopy
Strain FGY135 carrying plasmid pJOD010 (PGAL-GAP1-GFP) was cotransformed with plasmids pJK64, pJK65, or pRS317 (vector without insert). Cells were grown in allantoin media with 2% raffinose and 0.2% glucose to an OD600 of 23, and subsequently suspended in allantoin media with 4% galactose and 1 µg ml1 DAPI for 5 h. Cells were suspended in media containing 0.4% low melting point agar (37°C) and quickly mounted on slides for microscopic observation. Live cells were viewed at RT using an Axiophot microscope (Carl Zeiss MicroImaging, Inc.) with a Plan-Apochromat 63x/1.40 objective. Digital images of cells examined using Nomarski optics, and of the fluorescence associated with GFP and DAPI (standard filter sets), were captured using a C4742-95 CCD camera (Hamamatsu Photonics) and QED Imaging software (Media Cybernetics). Image files were incorporated into figures using Photoshop CS (Adobe Systems, Inc.).
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Acknowledgments |
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This work was supported by the Ludwig Institute for Cancer Research and a grant from the EU (EFFEXPORT project; QLK3-CT-2001-00533).
Submitted: 18 August 2004
Accepted: 4 November 2004
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References |
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