Correspondence to Matthias Seedorf: m.seedorf{at}zmbh.uni-heidelberg.de
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Introduction |
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In contrast to transport by the so-called classical SEC pathway via the ER, Golgi apparatus, and secretory vesicles, we have shown that the yeast integral membrane protein Ist2p reaches the plasma membrane independently of the formation of COPII-coated vesicles. The transport of Ist2p does not depend on Sec12p and Sec23p, on the transport of vesicles along actin filaments (which is mediated by Myo2p), on the formation of vesicles at the Golgi (which is mediated by Sec7p), or on the Sec1p-dependent fusion of vesicles with the plasma membrane (Jüschke et al., 2004). These observations have led to the hypothesis that a connection between the localization of IST2 mRNA and the unusual trafficking of the protein could exist (Jüschke et al., 2004). IST2 mRNA belongs to a group of transcripts that accumulate at the cortex of daughter cells (Takizawa et al., 2000; Shepard et al., 2003). These mRNAs interact with the RNA-binding protein She2p, which connects mRNA particles with the myosin motor Myo4p via the She3p adaptor and, thereby, mediates the translocation of the RNA along the polarized actin cytoskeleton into the daughter cell (Gonsalvez et al., 2005).
The transport of IST2 mRNA by the She machinery is required for the expression of Ist2p in the plasma membranes of daughter cells (Takizawa et al., 2000; Jüschke et al., 2004). The observed ablation of Ist2p expression in small and medium-sized daughter cells in she mutants could be explained by a lack of transport and synthesis of Ist2p into daughter cells. This is why, in combination with the diffusion barrier for integral plasma membrane proteins located at the bud-neck region of the plasma membrane, she
mutants that fail to transport IST2 mRNA into daughter cells lack Ist2p in their plasma membranes (Takizawa et al., 2000; Jüschke et al., 2004). These observations suggest that Ist2p is synthesized at the cortical ER and that daughter cells need the transport of RNA for local synthesis. However, the expression of Ist2p in mother cells does not require the function of the She machinery; therefore, She-mediated mRNA transport is not a general prerequisite for Ist2p synthesis.
Ist2p is predicted to have eight TM segments with NH2 and COOH termini oriented to the cytosol. In this study, we have identified the segment encoding the COOH-terminal domain as the sorting determinant, which is able to direct Ist2p and other membrane proteins via a novel pathway through the cortical ER to the plasma membrane. We suggest that this pathway involves a spatial control of IST2 translation, a local insertion of the newly synthesized protein into specific domains of the ER membrane, and the transport of Ist2p by a novel (SEC independent) mechanism to the plasma membrane.
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Results |
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To determine whether Prm1-GFP-Ist2C passes through the ER, we compared the apparent molecular mass of Prm1-CFP and Prm1-GFP-Ist2C with that of the calculated molecular mass. The modification of 14 predicted consensus sites for N-linked glycosylation should lead to a decreased mobility of the protein in SDS-PAGE and would indicate a passage through the ER. Prm1-CFP showed a major band of 115 kD with a faint, diffuse smear above it, whereas Prm1-GFP-Ist2C migrated as a band of 180 kD (Fig. 3 B). The treatment of membranes from these cells with peptide N-glycosidase F, an enzyme that removes N-linked sugar moieties, shifted both Prm1-CFP and Prm1-GFP-Ist2C bands into faster migrating species of 95 and 150 kD, respectively, indicating that both proteins had received N-linked core glycosylation at the ER. These results demonstrate that adding Ist2C to the COOH terminus of a membrane protein does not prevent its trafficking through the ER nor prevents its accessibility to the core glycosylation machinery.
Trafficking of Ist2p through the Golgi apparatus
To determine whether Ist2p is directly transferred from the ER to the plasma membrane or if the trafficking of the protein involves passage through the Golgi apparatus, we investigated whether the N-linked glycosylation sites receive Golgi-specific mannose modifications. Modifications of N-linked oligosaccharides in the yeast Golgi complex is initiated by the transfer of a mannose residue to the core oligosaccharide in an -1,6-linkage (Nakayama et al., 1992). This modification is followed by further heterogeneous elongation and branching, resulting in a final addition of
-1,3-linked mannose residues to the branched chain (Raschke et al., 1973). These reactions are initiated in distinct compartments of the Golgi complex:
-1,6-linkage occurs at the cis-Golgi, and
-1,3-linkage occurs at the medial- and trans-Golgi (Brigance et al., 2000).
We introduced constructs encoding Prm1-GFP-Ist2C and Prm1-CFP in MATa and MAT strains and induced the expression by mixing the cell cultures of opposite mating types. 75 min after induction, Prm1-CFP and Prm1-GFP-Ist2C were immunoprecipitated with GFP-specific antibodies, and the isolated proteins were probed with an antibody recognizing GFP to determine the recovery of the proteins. Prm1-CFP was seen as a major 115-kD band with some additional faint, diffuse bands that had reduced mobility (Fig. 4, lane 1). These diffuse bands were also recognized by antibodies specific for
-1,6- or
-1,3-mannose modifications (Fig. 4, lanes 2 and 3), indicating that only a minor part of Prm1-CFP reached the cis- and trans-Golgi compartments at the time of induction.
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To further investigate the trafficking of Ist2p through the Golgi, we chose the chloride channel protein Gef1p as another reporter protein. Gef1p is processed during its transport in the TGN by the furin protease Kex2p, which recognizes amino acid KR at positions 136 and 137 as cleavage sites in Gef1p (Fig. 5 A; Wachter and Schwappach, 2005). This processing allows us to monitor passage through the TGN. To determine whether Gef1-GFP-Ist2C was transported through late Golgi cisternae, we investigated its processing by Kex2p protease. Gef1-GFP, with the Kex2p cleavage site deleted (KR to AA mutation, Gef1(KR>AA)-GFP), migrated as a 110-kD band, whereas the majority of wild-type Gef1-GFP was cleaved into a 90-kD band (Fig. 5 B, first and second lanes). This processing by Kex2p was not observed in cells expressing Ist2C-tagged Gef1-GFP. Gef1-GFP-Ist2C and Gef1(KR>AA)-GFP-Ist2C migrated as bands of identical size (Fig. 5 B, third and fourth lanes). These data indicate that the COOH-terminal domain of Ist2p prevents the transport of fusion proteins through the TGN and is consistent with the bypassing of the medial- and trans-Golgi, as shown in Fig. 4 (lanes 46).
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The dimerization allowed us to study the sorting function of Ist2C in trans by coexpressing a wild-type as well as an Ist2C-tagged Gef1p. Diploids that coexpressed Gef1-RFP and Gef1-GFP-Ist2C showed a diminished dotlike, intracellular localization of Gef1-RFP, which partially overlapped with Gef1-GFP-Ist2C at the cell periphery (Fig. 5 C). These observations indicate that both subunits assemble and that Ist2C functions in trans as a dominant plasma membrane sorting signal for the dimer. We took advantage of this fact and analyzed the Kex2p cleavage of a protein C (PC) epitopetagged version of Gef1p (Gef1-4PC) in cells that coexpress either Gef1-GFP or Gef1-GFP-Ist2C. In a situation that led to the homodimerization of Gef1-4PC or to the heterodimerization of Gef1-4PC with Gef1-GFP, the majority of Gef1-4PC migrated as the processed form (Fig. 5 D, first lane). In the case that Gef1-4PC formed a heterodimer with Gef1-GFP-Ist2C, a significant portion of the Gef1-4PC was shifted into a slower migrating species of the same size as Gef1(KR>AA)-4PC (Fig. 5 D, second and fourth lanes). This means that the presence of one copy of Ist2C targets the dimer from the ER to the plasma membrane and prevents the wild-type subunit from being cleaved, which suggests that this transport occurs without passing through the Kex2p-positive TGN compartment. This is consistent with the previously observed bypassing of the medial- and trans-Golgi compartment.
The COOH-terminal domain of Ist2p directs proteins independently of COPII-mediated vesicular transport to the plasma membrane
Because Ist2p trafficking occurs independently of SEC12, SEC23, SEC7, and SEC1-mediated transport (Jüschke et al., 2004), we asked if adding Ist2C to membrane proteins is sufficient to bypass the classical SEC pathway to the plasma membrane. To investigate this question, we chose Ste6p, the a-factor pheromone transporter and member of the ATP-binding cassette superfamily, because its membrane topology has been well established by gene fusion experiments (Geller et al., 1996). We created a fusion protein of yEmCitrine, an improved YFP variant, with the first two TM segments of Ste6p (YFP-Ste6TM1 + 2) and expressed this protein under the control of the GAL1 promoter (Fig. 6). According to the topology of Ste6p, the YFP-Ste6TM1 + 2 chimera should result in a membrane protein with both NH2 and COOH termini facing the cytosol (Geller et al., 1996). The expression of YFP-Ste6TM1 + 2 without an additional moiety at its COOH terminus resulted in the accumulation in an ER-associated compartment (unpublished data). This compartment has recently been described as a quality control subcompartment of the ER (Huyer et al., 2004).
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SEC18-dependent vesicular fusion is not required for the sorting of Ist2p to the plasma membrane
The vesicle-mediated transport steps of the SEC pathway are mediated by the SNARE-dependent fusion of donor and target membranes (Rothman and Wieland, 1996). SNARE molecules, which are located on opposite membranes, form stable four-helix bundles and, thereby, induce membrane fusion. For membrane fusion to occur continuously, all of these reactions depend on the regeneration of separate SNARE molecules, a process that is catalyzed by the activity of an AAA-ATPase. In yeast, this enzyme is encoded by SEC18, the orthologue of NSF in mammalian cells (Sollner et al., 1993). In the yeast sec18-1 mutant protein, transport ceases almost immediately after shifting the cells to the nonpermissive growth temperature of 37°C (Graham and Emr, 1991). Therefore, this mutant could be used to analyze whether trafficking on the Ist2 pathway involves classic membrane fusion events. To investigate the trafficking of newly synthesized Prm1-GFP-Ist2C, we induced its expression in a sec18-1 MATa strain by adding prewarmed media containing -factor. These cells were incubated for another 60 min at 37°C. Although the expression of Prm1-GFP-Ist2C was low, some of the protein appeared in a peripheral patchlike staining (Fig. 7 A), which suggests sorting to the plasma membrane. To further test whether the newly synthesized Prm1-GFP-Ist2C had reached the plasma membrane, we investigated its accessibility for protease digestion from the outside. We used the protease trypsin instead of pronase because sec18 mutants have a weak cell wall at nonpermissive conditions that is even further weakened by the initiation of the mating response. We also coexpressed Dpm1-CFP to test the intactness of the plasma membrane after protease addition. This ER membrane protein has one COOH-terminally located TM segment and a large NH2 terminus, which is exposed to the cytosol (Faulhammer et al., 2005) and would be digested in the case of protease entering the cytosol. The trypsin resistance of Dpm1-CFP (Fig. 7 B, lanes 69) indicates that the plasma membrane has remained intact during the protease treatment. The occurrence of a 28-kD breakdown product of Dpm1-CFP after the mechanical disruption of the plasma membrane confirmed the trypsin sensitivity of Dpm1-CFP (Fig. 7 B, lane 1). Adding increasing amounts of trypsin protease resulted in the cleavage of the 180-kD band of Prm1-GFP-Ist2C into faster migrating bands (Fig. 7 B, lane 9). Under the same conditions, Prm1-CFP remained intact (Fig. 7 B, lanes 25). These results indicate that Ist2C-tagged Prm1p was located at the plasma membrane under conditions in which the forward transport of Prm1-CFP was abolished. Altogether, these results show that the transport of newly synthesized Prm1-GFP-Ist2C to the plasma membrane does not require vesicular fusion events that depend on Sec18p function.
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Together, these results demonstrate that Ist2C-tagged Prm1p at the ER can enter two different routes: a SEC-independent transport, which delivers the protein to the plasma membrane, or the ER to Golgi step of the classical SEC-dependent transport route, which results in trafficking to the cis-Golgi.
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Discussion |
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Ist2C-tagged proteins pass through the ER on their way to the plasma membrane, as shown by the ER core glycosylation of Ist2C-tagged Prm1p. After insertion into the ER membrane, Ist2p can enter two different routes. One leads independently of the function of Sec12p and Sec18p to the plasma membrane, indicating that this pathway operates without the function of the COPII coat and SNARE-mediated vesicular fusion events. To our knowledge, this is the first example of such a pathway for an integral membrane protein. The selective secretion in the absence of the COPII components Sec13p and Sec24p has recently been observed for Hsp150p (Fatal et al., 2002, 2004). However, in contrast to the transport of Ist2p, the secretion of Hsp150p depends on Sec23p and Sec12p function (Fatal et al., 2002). The other route leads to SEC18-dependent transport Ist2p to the cis-Golgi. Although our immunoprecipitation assays are not quantitative, we suggest that only a small fraction of Ist2C-tagged Prm1 reporter protein reaches the cis-Golgi. The extreme COOH terminus of Ist2p, KKKL, comprises a strong KKXX ER-retrieval signal that could very well initiate the retrograde transport of Ist2p back to the ER. From there, the protein might get another chance to enter ER domains, which are capable of SEC-independent sorting to the plasma membrane. Whether this transport through the classical SEC pathway to the cis-Golgi and subsequent modification by the addition of sugar side chains are necessary for the function of proteins on this route is still unknown. The transport via the classical SEC pathway could simply represent the misincorporation of Ist2p into COPII-coated vesicles at the ER. The bypassing of late Golgi compartments has been shown by the lack of -1,3-mannose modification of Prm1-GFP-IST2C and is further supported by the observed lack of Kex2p cleavage in the Ist2C-tagged Gef1 reporter protein.
The simplest model that explains how the sorting of Ist2p could operate would be a mechanism that includes a local translation of IST2 mRNA at cortical ER sites, which are competent to initiate the SEC18-independent transport to the plasma membrane. Information within the mRNA, which encodes the COOH-terminal domain of Ist2p, could spatially restrict the translation and, thereby, direct the insertion of the nascent polypeptide chain into the cortical ER. As shown for many localized mRNAs, IST2 mRNA is present as an RNP particle, which is exported from the nucleus into the cytosol. According to the current model of RNA transport in yeast, the translation of the transported mRNAs is repressed by cis-acting, RNA localization elements, which have been predicted to form stem loops (Chartrand et al., 1999, 2002). In the right environment and at the cortical ER, the translational repression is released, and the newly synthesized protein is inserted into the cortical ER membrane. The localization of IST2 mRNA to the cortex of daughter cells by the She machinery is not necessary for its translation, indicating that Ist2p could be synthesized at the cortical ER in daughter and mother cells (Takizawa et al., 2000; Jüschke et al., 2004). Other components that are distinct from the She proteins, which are present in the IST2 mRNA particle, might regulate this local translation. The candidates are RNA-binding proteins (e.g., Khd1p or Scp160p), which repress the translation of ASH1 mRNA (Irie et al., 2002). The postulated local translation of Ist2C-tagged proteins at the cortical ER does not lead to a spatial restriction of trafficking through the confined areas of the ER. Ist2C-tagged proteins have access to other proteins that are sorted via the classical SEC pathway, as shown by the function of Ist2C as a sorting determinant in trans and by the cis-Golgi modification of Ist2C-tagged Prm1p.
In contrast to a model based on a locally restricted translation of IST2 mRNA, the translation and insertion of the polypeptide could occur randomly at ER membranes. In this case, strong proteinacious sorting signals in Ist2C would confer an efficient, posttranslational recruitment of Ist2p into COPII-independent, ER exit sites. Because of the time required for the folding of GFP, we cannot exclude this possibility. The observed function of Ist2C as a sorting determinant in trans rules out a third mechanism; namely, that the protein would be extracted from the ER into the cytosol before insertion into the plasma membrane.
To explain the transport from the cortical ER to the neighboring plasma membrane, we suggest two possibilities: a local, transient fusion of part of the cortical ER with the plasma membrane or a fission and fusion mechanism between the cortical ER and the plasma membrane with a novel type of Ist2p containers. The fusion of parts of the ER with the plasma membrane has been suggested to play a role in the process of rapid membrane expansion in macrophages during the formation of phagocytic cups, when macrophages engulf large pathogens (Gagnon et al., 2002). It has been proposed that the exocyst complex provides a direct contact between parts of the ER and the plasma membrane (Lipschutz et al., 2003; Toikkanen et al., 2003). This is supported by findings in yeast, in which a direct contact between translocon and exocyst components has been reported (Toikkanen et al., 2003), and by contacts between these membranes in neurons during the trafficking of N-methyl-D-aspartate receptors in synapses (Sans et al., 2003). The coupling of Ca2+ signaling between the plasma membrane and the sarcoplasmic reticulum in muscle cells (for review see Blaustein et al., 2002) and the transport of lipids from the cortical ER to the yeast plasma membrane (Pichler et al., 2001) are further examples of a close contact between the domains of the ER and plasma membrane.
To summarize, our data suggest that trafficking of an integral membrane protein by a novel pathway through the cortical ER operates independently of Sec12p- and Sec18p-mediated vesicle formation and fusion. Furthermore, we have identified a novel dominant sorting determinant that redirects membrane proteins on this route to the plasma membrane and that could, in this respect, serve as a tool for investigating intracellular membrane proteins.
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Materials and methods |
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Construction of plasmids
The plasmid pCJ083 encoding GFP-Ist2, which is under the control of its own promoter for integration into the LEU2 locus, and the plasmid pCJ070 encoding Hxt1-CFP, which is under the control of the GAL1 promoter for integration into the TRP1 locus, have been described previously (Jüschke et al., 2004). The plasmids pCJ097, pCJ099, pCJ100, and pCJ102 were derived from pCJ083 by replacing the full-length IST2 ORF with different versions: the sequence that encodes the COOH-terminal aa 455 of Ist2p (including the last two TM segments) and the COOH-terminal aa 358 of Ist2p, together with 995 nucleotides of the IST2 3'-untranslated region (UTR), were amplified and subcloned into the pCRII-TOPO vector (Invitrogen). Each fragment was introduced between the BamHI and XhoI sites of pCJ083, resulting in pCJ097 and pCJ099, respectively. The plasmid pCJ100 was created by ligating full-length IST2 together with 995 nucleotides of the IST2 3'-UTR into the BamHI and XhoI sites of pCJ083. The plasmid pCJ102 was made by introducing the sequence coding for the first 591 NH2-terminal amino acids of Ist2p together with 995 nucleotides of the IST2 3'-UTR into the BamHI and XhoI sites of pCJ083. The plasmid pCJ113 encoded yEmCitrine (Griesbeck et al., 2001) under the control of the GAL1 promoter for integration into the URA3 locus and was constructed by subcloning the GAL1 promoter and the yEmCitrine fragment of pKT211 into the SacI and BamHI sites of pRS306 (Sikorski and Hieter, 1989). The plasmids pCJ115 and pCJ119 were generated by introducing full-length IST2 or the sequence coding for the COOH-terminal aa 455, including the two last TM segments, into the BamHI and XhoI sites of pCJ113. The plasmids pCJ116 and pCJ124 were created by introducing sequences, which encode either an NH2-terminal fragment of Ste6p (aa 1109) fused to a COOH-terminal fragment of Ist2p (aa 592946) or an NH2-terminal fragment of Ste6p (aa 1109) fused to the mature part of Suc2p (aa 20532), into the BamHI and XhoI sites of pCJ113.
The plasmid pCJ137 encoding Prm1-GFP-Ist2C under the control of the endogenous PRM1 promoter for integration into the HIS3 locus was constructed by amplifying the 387 to 1983 nucleotide region of PRM1, which introduced a SacI and an XmaI restriction site. GFP-Ist2C was amplified from pCJ099, introducing an XmaI and an XhoI site. Both fragments were immediately ligated into the SacI and XhoI sites of pRS303 (Sikorski and Hieter, 1989).
The construction of plasmids encoding the GEF1 gene, a GFP-tagged version, and a four time PC epitopetagged version of the GEF1 gene (which are under the control of the MET25 promoter) and the mutagenesis of KR at position aa 136 and 137 of Gef1p to AA have been described previously (Wachter and Schwappach, 2005). The plasmid pMS470 was generated by exchanging GFP with RFP (tdimer2[12]; Campbell et al., 2002). The plasmid pMS471 was created by exchanging a fragment encoding GFP with a NotIXhoI fragment encoding GFP and the COOH-terminal domain of Ist2p (aa 592946).
Fluorescence microscopy
Yeast cells expressing GFP fusion proteins were analyzed as previously described (Jüschke et al., 2004). The cells were mounted in growth medium at room temperature and were examined live using an inverted microscope (model DM IRE2; Leica) with a 100x/1.4-0.7 oil immersion objective (model HCX PL APO CS; Leica). Images were acquired using a camera (model ORCA-ER CCD; Hamamatsu) controlled by the OpenLab software package (Improvision) and were processed with Adobe Photoshop.
Western blotting and susceptibility to external proteases
Expression of Prm1-GFP-Ist2C in sec18-1 cells (JY43) was induced at 37°C by the addition of 1/500 vol of 5 mg/ml -factor (T6901; Sigma-Aldrich) in DMSO. Western blotting using GFP- (1:20,000 diluted; provided by D. Görlich, University of Heidelberg, Heidelberg, Germany), Sec61p- (1:10,000 diluted),
-1,6-, and
-1,3-mannose (1:20,000 diluted; provided by A. Spang, Friedrich Miescher Laboratorium der Max Planck Gesellschaft, Tübingen, Germany) or 250 ng/ml PC-specific antibodies (Roche) was performed as described previously, as was the susceptibility of plasma membrane proteins to external proteases (Jüschke et al., 2004). As an alternative to pronase, we used trypsin for sec18-1 strains.
Immunoprecipitation
Strains of opposite mating types that expressed Prm1-CFP and Prm1-GFP-Ist2C were grown at 25°C in YEPD media (1% wt/vol yeast extract, 2% wt/vol bacto-peptone, and 2% wt/vol dextrose) to 1 OD600, and an equal volume of media with a temperature of 25 or 40°C was added. The cells were incubated for an additional 5 min at 25 or 33°C before cells of opposite mating types were mixed to induce the expression of Prm1p. 100 OD600 cells were harvested, and the resulting cell pellet was disrupted by vortexing for 5 min with 1 vol of glass beads and 2 vol of low salt buffer (20 mM Hepes-KOH, pH 7.6, 100 mM KOAc, 5 mM Mg(OAc)2, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, and complete protease inhibitor mix) according to the manufacturer's instructions (Roche). The lysate was then cleared of unbroken cells by centrifugation (1,200 g at 4°C for 2 min) and were subjected to centrifugation at 25,000 g at 4°C for 20 min. Membranes were resuspended in 1% (vol/vol) Triton X-100, 400 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and complete protease inhibitor mix (Roche) and were subjected to centrifugation at 25,000 g at 4°C for 20 min. The supernatant was incubated for 90 min at 4°C with protA beads (Amersham Biosciences), which were preloaded with 5 µl of affinity-purified, GFP-specific antibody and were washed four times for 5 min with 1 ml of buffer, followed by one wash with PBS (150 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4). To remove bound protein, the beads were incubated for 10 min at 50°C in 100 µl of high urea buffer (8 M urea, 5% [wt/vol] SDS, 200 mM Tris-HCl, pH 6.8, 1 mM EDTA, 0.05% [wt/vol] bromphenol blue, 5% [vol/vol] ß-mercaptoethanol, and 100 mM DTT), and 20 µl/lane were separated by 6% SDS-PAGE.
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Acknowledgments |
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This work was supported by a grant from the Deutsche Forschungsgemeinschaft (GK230/3).
Submitted: 7 March 2005
Accepted: 15 April 2005
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References |
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---|
Antonny, B., and R. Schekman. 2001. ER export: public transportation by the COPII coach. Curr. Opin. Cell Biol. 13:438443.[CrossRef][Medline]
Barlowe, C., and R. Schekman. 1993. SEC12 encodes a guanine-nucleotide-exchange factor essential for transport vesicle budding from the ER. Nature. 365:347349.[CrossRef][Medline]
Blaustein, M.P., V.A. Golovina, H. Song, J. Choate, L. Lencesova, S.W. Robinson, and W.G. Wier. 2002. Organization of Ca2+ stores in vascular smooth muscle: functional implications. Novartis Found. Symp. 246:125137; discussion 137141, 221227.[Medline]
Brachmann, C.B., A. Davies, G.J. Cost, E. Caputo, J. Li, P. Hieter, and J.D. Boeke. 1998. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast. 14:115132.[CrossRef][Medline]
Brigance, W.T., C. Barlowe, and T.R. Graham. 2000. Organization of the yeast Golgi complex into at least four functionally distinct compartments. Mol. Biol. Cell. 11:171182.
Campbell, R.E., O. Tour, A.E. Palmer, P.A. Steinbach, G.S. Baird, D.A. Zacharias, and R.Y. Tsien. 2002. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA. 99:78777882.
Chartrand, P., X.H. Meng, R.H. Singer, and R.M. Long. 1999. Structural elements required for the localization of ASH1 mRNA and of a green fluorescent protein reporter particle in vivo. Curr. Biol. 9:333336.[CrossRef][Medline]
Chartrand, P., X.H. Meng, S. Huttelmaier, D. Donato, and R.H. Singer. 2002. Asymmetric sorting of ash1p in yeast results from inhibition of translation by localization elements in the mRNA. Mol. Cell. 10:13191330.[CrossRef][Medline]
Cosson, P., and F. Letourneur. 1994. Coatomer interaction with di-lysine endoplasmic reticulum retention motifs. Science. 263:16291631.[Medline]
Dutzler, R., E.B. Campbell, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity. Nature. 415:287294.[CrossRef][Medline]
Fatal, N., T. Suntio, and M. Makarow. 2002. Selective protein exit from yeast endoplasmic reticulum in absence of functional COPII coat component Sec13p. Mol. Biol. Cell. 13:41304140.
Fatal, N., L. Karhinen, E. Jokitalo, and M. Makarow. 2004. Active and specific recruitment of a soluble cargo protein for endoplasmic reticulum exit in the absence of functional COPII component Sec24p. J. Cell Sci. 117:16651673.
Faulhammer, F., G. Konrad, B. Brankatschk, S. Tahirovic, A. Knodler, and P. Mayinger. 2005. Cell growthdependent coordination of lipid signaling and glycosylation is mediated by interactions between Sac1p and Dpm1p. J. Cell Biol. 168:185191.
Foti, M., A. Audhya, and S.D. Emr. 2001. Sac1 lipid phosphatase and Stt4 phosphatidylinositol 4-kinase regulate a pool of phosphatidylinositol 4-phosphate that functions in the control of the actin cytoskeleton and vacuole morphology. Mol. Biol. Cell. 12:23962411.
Gagnon, E., S. Duclos, C. Rondeau, E. Chevet, P.H. Cameron, O. Steele-Mortimer, J. Paiement, J.J. Bergeron, and M. Desjardins. 2002. Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell. 110:119131.[CrossRef][Medline]
Geller, D., D. Taglicht, R. Edgar, A. Tam, O. Pines, S. Michaelis, and E. Bibi. 1996. Comparative topology studies in Saccharomyces cerevisiae and in Escherichia coli. The N-terminal half of the yeast ABC protein Ste6. J. Biol. Chem. 271:1374613753.
Gietz, R.D., and R.A. Woods. 2002. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350:8796.[CrossRef][Medline]
Gonsalvez, G.B., C.R. Urbinati, and R.M. Long. 2005. RNA localization in yeast: moving towards a mechanism. Biol. Cell. 97:7586.[CrossRef][Medline]
Graham, T.R., and S.D. Emr. 1991. Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant. J. Cell Biol. 114:207218.[Abstract]
Griesbeck, O., G.S. Baird, R.E. Campbell, D.A. Zacharias, and R.Y. Tsien. 2001. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276:2918829194.
Heese-Peck, A., H. Pichler, B. Zanolari, R. Watanabe, G. Daum, and H. Riezman. 2002. Multiple functions of sterols in yeast endocytosis. Mol. Biol. Cell. 13:26642680.
Heiman, M.G., and P. Walter. 2000. Prm1p, a pheromone-regulated multispanning membrane protein, facilitates plasma membrane fusion during yeast mating. J. Cell Biol. 151:719730.
Huyer, G., G.L. Longsworth, D.L. Mason, M.P. Mallampalli, J.M. McCaffery, R.L. Wright, and S. Michaelis. 2004. A striking quality control subcompartment in Saccharomyces cerevisiae: the endoplasmic reticulum-associated compartment. Mol. Biol. Cell. 15:908921.
Irie, K., T. Tadauchi, P.A. Takizawa, R.D. Vale, K. Matsumoto, and I. Herskowitz. 2002. The Khd1 protein, which has three KH RNA-binding motifs, is required for proper localization of ASH1 mRNA in yeast. EMBO J. 21:11581167.
Jüschke, C., D. Ferring, R.P. Jansen, and M. Seedorf. 2004. A novel transport pathway for a yeast plasma membrane protein encoded by a localized mRNA. Curr. Biol. 14:406411.[CrossRef][Medline]
Konrad, G., T. Schlecker, F. Faulhammer, and P. Mayinger. 2002. Retention of the yeast Sac1p phosphatase in the endoplasmic reticulum causes distinct changes in cellular phosphoinositide levels and stimulates microsomal ATP transport. J. Biol. Chem. 277:1054710554.
Lipschutz, J.H., V.R. Lingappa, and K.E. Mostov. 2003. The excocyst affects protein synthesis by acting on the translocation machinery of the endoplasmic reticulum. J. Biol. Chem. 278:2095420960.
Middleton, R.E., D.J. Pheasant, and C. Miller. 1996. Homodimeric architecture of a ClC-type chloride ion channel. Nature. 383:337340.[CrossRef][Medline]
Muniz, M., P. Morsomme, and H. Riezman. 2001. Protein sorting upon exit from the endoplasmic reticulum. Cell. 104:313320.[CrossRef][Medline]
Munn, A.L., A. Heese-Peck, B.J. Stevenson, H. Pichler, and H. Riezman. 1999. Specific sterols required for the internalization step of endocytosis in yeast. Mol. Biol. Cell. 10:39433957.
Munro, S. 2004. Organelle identity and the organization of membrane traffic. Nat. Cell Biol. 6:469472.[CrossRef][Medline]
Nakayama, K., T. Nagasu, Y. Shimma, J. Kuromitsu, and Y. Jigami. 1992. OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharides. EMBO J. 11:25112519.[Abstract]
Pichler, H., B. Gaigg, C. Hrastnik, G. Achleitner, S.D. Kohlwein, G. Zellnig, A. Perktold, and G. Daum. 2001. A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids. Eur. J. Biochem. 268:23512361.
Raschke, W.C., K.A. Kern, C. Antalis, and C.E. Ballou. 1973. Genetic control of yeast mannan structure. Isolation and characterization of mannan mutants. J. Biol. Chem. 248:46604666.
Rayner, J.C., and H.R. Pelham. 1997. Transmembrane domain-dependent sorting of proteins to the ER and plasma membrane in yeast. EMBO J. 16:18321841.[Abstract]
Rossanese, O.W., J. Soderholm, B.J. Bevis, I.B. Sears, J. O'Connor, E.K. Williamson, and B.S. Glick. 1999. Golgi structure correlates with transitional endoplasmic reticulum organization in Pichia pastoris and Saccharomyces cerevisiae. J. Cell Biol. 145:6981.
Rothman, J.E., and F.T. Wieland. 1996. Protein sorting by transport vesicles. Science. 272:227234.[Abstract]
Sans, N., K. Prybylowski, R.S. Petralia, K. Chang, Y.X. Wang, C. Racca, S. Vicini, and R.J. Wenthold. 2003. NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex. Nat. Cell Biol. 5:520530.[CrossRef][Medline]
Sato, K., M. Sato, and A. Nakano. 2003. Rer1p, a retrieval receptor for ER membrane proteins, recognizes transmembrane domains in multiple modes. Mol. Biol. Cell. 14:36053616.
Schwappach, B., S. Stobrawa, M. Hechenberger, K. Steinmeyer, and T.J. Jentsch. 1998. Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p. J. Biol. Chem. 273:1511015118.
Shepard, K.A., A.P. Gerber, A. Jambhekar, P.A. Takizawa, P.O. Brown, D. Herschlag, J.L. DeRisi, and R.D. Vale. 2003. Widespread cytoplasmic mRNA transport in yeast: identification of 22 bud-localized transcripts using DNA microarray analysis. Proc. Natl. Acad. Sci. USA. 100:1142911434.
Sherman, F. 2002. Getting started with yeast. Methods Enzymol. 350:341.[Medline]
Sikorski, R.S., and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122:1927.
Sollner, T., M.K. Bennett, S.W. Whiteheart, R.H. Scheller, and J.E. Rothman. 1993. A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell. 75:409418.[CrossRef][Medline]
Takizawa, P.A., J.L. DeRisi, J.E. Wilhelm, and R.D. Vale. 2000. Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier. Science. 290:341344.
Thomas, B.J., and R. Rothstein. 1989. Elevated recombination rates in transcriptionally active DNA. Cell. 56:619630.[CrossRef][Medline]
Toikkanen, J.H., K.J. Miller, H. Soderlund, J. Jantti, and S. Keranen. 2003. The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae. J. Biol. Chem. 278:2094620953.
Wachter, A., and B. Schwappach. 2005. The yeast CLC chloride channel is proteolytically processed by the furin-like protease Kex2p in the first extracellular loop. FEBS Lett. 579:11491153.[CrossRef][Medline]
Watanabe, R., and H. Riezman. 2004. Differential ER exit in yeast and mammalian cells. Curr. Opin. Cell Biol. 16:350355.[CrossRef][Medline]