Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat
2011; Springer Nature; Volume: 31; Issue: 4 Linguagem: Inglês
10.1038/emboj.2011.444
ISSN1460-2075
AutoresLeslie Kung, Silvère Pagant, Eugene Futai, Jennifer G. D’Arcangelo, Roy Buchanan, John Dittmar, Robert J.D. Reid, Rodney Rothstein, Susan Hamamoto, Erik L. Snapp, Randy Schekman, Elizabeth A. Miller,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle9 December 2011free access Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat Leslie F Kung Leslie F Kung Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Silvere Pagant Silvere Pagant Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Eugene Futai Eugene Futai Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Jennifer G D'Arcangelo Jennifer G D'Arcangelo Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Roy Buchanan Roy Buchanan Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author John C Dittmar John C Dittmar Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Robert J D Reid Robert J D Reid Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Rodney Rothstein Rodney Rothstein Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Susan Hamamoto Susan Hamamoto Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Erik L Snapp Erik L Snapp Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, NY, USA Search for more papers by this author Randy Schekman Randy Schekman Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Elizabeth A Miller Corresponding Author Elizabeth A Miller Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Leslie F Kung Leslie F Kung Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Silvere Pagant Silvere Pagant Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Eugene Futai Eugene Futai Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Jennifer G D'Arcangelo Jennifer G D'Arcangelo Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Roy Buchanan Roy Buchanan Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author John C Dittmar John C Dittmar Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Robert J D Reid Robert J D Reid Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Rodney Rothstein Rodney Rothstein Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Susan Hamamoto Susan Hamamoto Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Erik L Snapp Erik L Snapp Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, NY, USA Search for more papers by this author Randy Schekman Randy Schekman Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA Search for more papers by this author Elizabeth A Miller Corresponding Author Elizabeth A Miller Department of Biological Sciences, Columbia University, New York, NY, USA Search for more papers by this author Author Information Leslie F Kung1,‡, Silvere Pagant1,‡, Eugene Futai2, Jennifer G D'Arcangelo1, Roy Buchanan1, John C Dittmar1, Robert J D Reid3, Rodney Rothstein3, Susan Hamamoto2, Erik L Snapp4, Randy Schekman2 and Elizabeth A Miller 1 1Department of Biological Sciences, Columbia University, New York, NY, USA 2Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA 3Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA 4Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, NY, USA ‡These authors contributed equally to this work *Corresponding author. Department of Biological Sciences, Columbia University, 1212 Amsterdam Ave MC2456, New York, NY 10027, USA. Tel.: +1 212 854 2264; Fax: +1 212 865 8246; E-mail: [email protected] The EMBO Journal (2012)31:1014-1027https://doi.org/10.1038/emboj.2011.444 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly. Introduction Protein traffic within the endomembrane system of eukaryotic cells occurs via transport vesicles that ferry various molecules between compartments. Vesicles are created by cytoplasmic coat proteins that perform two fundamental roles: selection of cargo molecules (and exclusion of residents) and transformation of the donor membrane into highly curved spherical structures (Kirchhausen, 2000; Stagg et al, 2007). Coat assembly is often driven by GTP, with a small monomeric GTPase acting as a regulator of coat assembly; in the GTP-bound state, the G-protein recruits additional cargo adaptor proteins and membrane scaffold proteins, which are subsequently released upon GTP hydrolysis. In this way, coat assembly, triggered by GTP binding, couples cargo recruitment with membrane deformation, then GTP hydrolysis permits uncoating to expose the fusion apparatus required for vesicle delivery to the target compartment (Bonifacino and Glick, 2004; Miller and Barlowe, 2010). Endoplasmic reticulum (ER)-derived transport vesicles are generated by the COPII coat, comprising five proteins that assemble on the cytoplasmic surface of the ER membrane (Barlowe et al, 1994). The GTPase, Sar1p, initiates assembly, recruiting the cargo adaptor platform, Sec23p/Sec24p, and the outer coat, Sec13p/Sec31p. In minimally reconstituted systems, these components assemble in a hierarchical manner, with each layer dependent on the previous one (Matsuoka et al, 1998; Antonny et al, 2001). Furthermore, each layer of the COPII coat contributes to the GTP cycle by stimulating the relatively poor GTPase activity of Sar1p. Sec23p is the GTPase-activating protein (GAP) for Sar1p (Yoshihisa et al, 1993), contributing catalytic residues to the hydrolysis reaction (Bi et al, 2002). Sec31p potentiates the action of Sec23p by optimally positioning the catalytic pocket (Antonny et al, 2001; Bi et al, 2007). Therefore, maximal GTPase activity is achieved only upon full coat assembly. On synthetic liposomes, coat assembly in the presence of GTP is remarkably transient since both the Sec23/24p and Sec13/31p layers have low affinity for Sar1p•GDP (Antonny et al, 2001). Thus, intrinsic GTPase regulation by coat proteins themselves creates a paradox: how is coat assembly stabilized for a sufficient amount of time to generate a vesicle when the fully assembled coat triggers its own disassembly? The existence of additional factors required in vivo for the negative regulation of Sar1p GTPase activity and/or stabilization of the COPII coat after GTP hydrolysis by Sar1p has long been postulated. Several lines of evidence point to Sec16p as a potential regulator of COPII vesicle biogenesis. Sec16p is essential for ER-to-Golgi transport in vivo (Kaiser and Schekman, 1990), is predominantly localized at ER exit sites and is important for their organization in Pichia pastoris, mammals and Drosophila (Watson et al, 2006; Bhattacharyya and Glick, 2007; Iinuma et al, 2007; Ivan et al, 2008; Hughes et al, 2009). Purified Sec16p is not strictly required for COPII vesicle formation from synthetic liposomes, but clearly stimulates this process (Matsuoka et al, 1998; Supek et al, 2002). Sec16p is large (∼240 kDa), forms oligomers and interacts with all COPII coat proteins (Espenshade et al, 1995; Gimeno et al, 1996; Shaywitz et al, 1997; Whittle and Schwartz, 2010). Taken together, these data suggest that Sec16p acts as a platform for COPII protein assembly that would nucleate oligomerization or organization of dispersed COPII subunits. The model that the stabilizing role of Sec16p on COPII assembly is structural rather than catalytic is sustained by the observation that purified Sec16p had no effect on Sar1p GTPase activity in vitro (Supek et al, 2002). In addition to functioning in simple binding and release of the COPII coat, the GTPase activity of Sar1p also appears to play a role in vesicle scission and cargo recruitment. Either deleting the N-terminal amphipathic α-helix of Sar1p or abrogating Sar1p GTPase activity causes defects in vesicle release, with spherical buds remaining attached to the donor membrane (Bielli et al, 2005; Lee et al, 2005). These observations suggest that the membrane curvature induced by the N-terminal helix (Lee et al, 2005), oligomerization of Sar1p (Long et al, 2010) and the lipid destabilization (Settles et al, 2010) that accompanies helix insertion and removal during the GTPase cycle are important for scission of the vesicle neck. The potential involvement of GTPase activity in cargo capture was suggested by single molecule fluorescence studies that showed improper clustering of the cargo protein, Bet1p, in the presence of a non-hydrolysable GTP analogue (Tabata et al, 2009). However, the mechanism by which GTP hydrolysis might impact the process of cargo capture in the context of a complex membrane remains unclear, in particular, in light of numerous experiments that have delineated Sec24p as the cargo-binding component of the COPII coat (Miller et al, 2002, 2003; Mossessova et al, 2003; Mancias and Goldberg, 2008). A combination of genetic, biochemical and structural analyses have clearly defined the cargo-binding function of Sec24p (Miller et al, 2003; Mossessova et al, 2003). In contrast, Sec24p had no influence on the GTP cycle of Sar1p when tested in a minimal system using only the purified COPII coat proteins (i.e., Sar1p, Sec23/24p and Sec13/31p; Bi et al, 2007), suggesting a function as a relatively inert platform that co-opts cargo via direct interaction with ER export signals on its various clients. In this study, we demonstrate that a fragment of Sec16p functions to negatively regulate the Sec23p- and Sec31p-stimulated GTPase cycle of Sar1p. We show that this activity is surprisingly dependent on Sec24p. Finally, we report that the effect of altering this regulation, via a new mutation in Sec24p, results primarily in the release of small COPII vesicles, suggesting a novel role for both Sec24p and Sec16p in regulating the GTP cycle of the COPII coat to prevent premature vesicle release. Results Three independent cargo-binding sites have been well defined on yeast Sec24p (Miller et al, 2003; Mossessova et al, 2003), and the identification of additional cargo-binding sites on mammalian isoforms of Sec24p (Farhan et al, 2007; Mancias and Goldberg, 2008) coupled with the observation that many yeast cargo proteins remain unaffected by mutation in the three yeast sites (Miller et al, 2003, 2005) raises the prospect that additional sites of cargo interaction remain to be identified. In searching for such sites, we employed an alanine-scanning mutagenesis approach to isolate novel surface mutations on Sec24p. One such mutant, termed sec24-m11, contained alterations in two adjacent acidic residues (E504 and D505) on a surface loop flanking the so-called ‘A-site’ or Sed5p-binding site (Figure 1A). This double mutant was temperature sensitive when present as the sole copy of Sec24p and was unable to support viability at any temperature in a sec24Δ iss1Δ double-mutant background, where the close Sec24p paralogue, Iss1p, was also deleted (Figure 1B). We tested the phenotypes of the single mutations, E504A or D505A, and detected no growth defects in either a sec24Δ or a sec24Δ iss1Δ background (EAM, unpublished observations), suggesting that preserving a single acidic residue suffices for viability. Figure 1.The m11 site defines a novel mutation on the surface of Sec24p. (A) The structure of Sar1p, Sec23p and Sec24p showing the positions of the known cargo-binding sites, the A-, B- and C-sites. A novel mutation, the m11 site, is located on a surface loop adjacent to the A-site. (B) The phenotype associated with the m11 mutation of Sec24p was assessed by introducing the mutant gene into strains containing chromosomal deletions in SEC24 and ISS1 as indicated and testing the ability of these strains to grow in the presence of 5-FOA, which counterselects for a plasmid-borne copy of SEC24. In a sec24Δ strain, sec24-m11 was able to confer viability at 30°C (left panel) but growth was significantly impaired at 38°C (middle panel), and in the context of a sec24Δ iss1Δ double null strain was unable to support viability (right panel). Download figure Download PowerPoint The m11 mutation in Sec24 impedes secretion in vivo and causes ER membrane accumulation We examined secretory pathway function in cells where the sole copy of Sec24p contained the E504A and D505A mutations. Both the GPI-anchored cell wall protein, Gas1p, and the soluble vacuolar hydrolase, CPY, were profoundly delayed in their maturation from precursor ER forms to Golgi-modified forms, suggesting a block in ER exit at restrictive temperature (Figure 2A). Protein biogenesis delays were also observed at lower temperature for the Sec24p-m11 mutant (30°C; Supplementary Figure S1A) and for the Sec24p-R230,235A B-site mutant that is defective in packaging the fusogenic SNARE, Bet1p, whereas the Sec24p-W897A A-site mutant showed no such defects (Supplementary Figure S1B). Since CPY and Gas1p are recruited into COPII vesicles by two independent cargo receptor systems, these observations were our first clue that the m11 mutation might represent a relatively severe lesion that impacts secretion in general as opposed to a cargo-specific defect. Indeed, live-cell imaging of sec24-m11 cells expressing GFP fused to the ER resident protein, Cyb5p, revealed a dramatic expansion of the cortical ER relative to that of wild-type cells, which showed predominantly perinuclear ER fluorescence (Figure 2B). Conversely, cells expressing either the A- or B-site mutant forms of Sec24p showed normal perinuclear ER (Supplementary Figure S1C). The proliferation of internal membranes was confirmed by electron microscopy: wild-type cells showed normal perinuclear ER with several short cortical strands (Figure 2C), whereas sec24-m11 cells accumulated abundant internal membranes, some of which were continuous with the perinuclear ER (Figure 2D). This phenotype is similar to early sec mutants that impair vesicle formation (Kaiser and Schekman, 1990). We consider it most likely that the m11 mutation exerts a partial and selective effect that does not induce global destabilization of Sec24p, in part because of the surface location of the lesion and in part because the mutant protein was readily expressed and purified in complex with Sec23p from yeast cells. Figure 2.Sec24p-m11 blocks secretion and causes accumulation of ER membranes. (A) Maturation of Gas1p or CPY was monitored by pulse-chase analysis after shift to 37°C. Gas1 underwent rapid conversion from the ER precursor (p) form to the Golgi-modified mature (m) form in wild-type cells, whereas this maturation was largely absent from cells expressing Sec24p-m11 as the sole copy of Sec24p. Similarly, CPY accumulated as its ER p1 form in Sec24p-m11 cells but matured normally to Golgi-modified p2 and vacuolar mature (m) forms in wild-type cells. (B) Live-cell images of wild-type (left panel) or sec24-m11 (centre and right panels) strains expressing the ER marker protein, GFP–Cyb5p, following shift to 37°C for 1 h prior to imaging. The sec24-m11 mutant strain exhibits proliferated internal membranes adjacent to the plasma membrane, consistent with accumulation of cortical ER. Scale bar is 5 μm. (C) TEM images of wild-type cells grown at 37°C reveal the endoplasmic reticulum surrounding the nucleus (Nuc) with small strands of cortical membrane underlying the plasma membrane (arrowheads). The vacuole (Vac) stains darkly. Scale bar is 500 nm. (D) TEM analysis of sec24-m11 cells shifted to 37°C for 1 h prior to fixation shows elaborated strands of membrane extending from the nucleus that completely fill the cortical areas of the cell adjacent to the plasma membrane (arrowheads), as well as large internal accumulations of membranes, consistent with a defect in the generation of COPII vesicles causing proliferation of the ER. Scale bar is 500 nm. Download figure Download PowerPoint Sec24p-m11 causes a GTP-dependent vesicle budding defect in vitro We tested the ability of the purified Sec23p/Sec24p-m11 complex to generate transport vesicles in vitro using an established reconstitution assay (Barlowe et al, 1994). Radiolabelled pre-pro-α-factor was translocated into microsomal membranes that were subsequently washed with urea to remove endogenous COPII proteins and then incubated with Sar1p, Sec13/31p and either wild-type Sec23/24p or Sec23p in complex with Sec24p-m11. Budding was initiated by the addition of guanine nucleotides (GDP, GTP or GMP-PNP) and the slowly sedimenting vesicle fraction was separated from the dense donor membranes by differential centrifugation. The amount of glycosylated pro-α-factor released into the vesicle fraction was quantified by Concanavalin A precipitation, and the budding efficiency calculated as the percentage of pro-α-factor in the vesicle fraction relative to that in the starting membranes. In the presence of the non-hydrolysable GTP analogue, GMP-PNP, Sec24p-m11 was able to generate COPII vesicles to a similar extent as the wild-type protein (Figure 3A); however, when GTP was included in the incubation, the budding efficiency of the Sec24p-m11 reaction was <50% of that containing wild-type Sec24p (Figure 3A). We note that the budding efficiency of wild-type reactions containing GTP is much greater than that with GMP-PNP (Figure 3A), a phenomenon that has been reported previously (Rexach and Schekman, 1991) and that likely stems from multiple rounds of coat binding, budding and release upon GTP hydrolysis. Similar experiments using a modified method that allows for immunoblotting of a range of specific cargo proteins showed similar effects: all cargo proteins that we examined were decreased in their abundance in the vesicle fraction generated by Sec24p-m11 in the presence of GTP but were relatively normal in the presence of GMP-PNP (EAM, unpublished observations). Furthermore, when Sec24p-m11 was used in a cargo-capture assay that generates pre-budding complexes, all cargoes were recruited with efficiency equal to that of wild-type Sec24p (Figure 3B), suggesting the m11 mutation does not correspond to a canonical cargo-binding motif but rather impacts vesicle formation more broadly. Thus, one explanation for a GTP-dependent budding defect is that the coat fails to turn over sufficiently to generate iterative rounds of budding. Figure 3.In-vitro defects in GTP-dependent vesicle formation are associated with the m11 mutation. (A) In-vitro formation of COPII vesicles was quantified by measuring release of radiolabelled pro-α-factor into a slowly sedimenting vesicle fraction following incubation at room temperature with the COPII coat supplemented with either wild-type Sec24p or Sec24p-m11. Reactions performed in the presence of GMP-PNP showed a subtle but significant difference between wild-type and m11 mutant reactions, whereas the presence of GTP caused a highly significant reduction in the budding of pro-α-factor in reactions containing Sec24p-m11. Error bars represent standard deviation from 4 to 6 independent experiments; statistical analysis was an unpaired t-test. (B) Cargo selection into pre-budding complexes was tested by incubating urea-washed microsomal membranes with GST–Sar1p and either wild-type Sec23/24p or Sec23/Sec24p-m11 as indicated. Membranes were solubilized and subjected to glutathione affinity precipitation followed by immunoblotting for the cargo proteins indicated. Sec24p-m11 permitted recruitment of all cargoes in a nucleotide-dependent manner. Tot represents 2% of the total microsomal membranes that initiated the reaction. (C) Coat assembly was assessed using a liposome flotation assay. COPII components were incubated with synthetic liposomes in the presence of wild-type Sec24p or Sec24p-m11 as indicated. Binding reactions were mixed with sucrose and overlaid with additional sucrose layers prior to centrifugation and collection of the floated material. Liposomes and bound proteins recovered from the top of the gradient were detected by SDS–PAGE and SYPRO red staining. The coat assembled normally with both wild-type and m11 forms of Sec24p, with stable recruitment only detected in the GMP-PNP reaction. (D) Coat assembly on the surface of synthetic liposomes was monitored in real time by light scattering. Recruitment of coat components to the surface of the liposome induces an increase in the light scattering signal. In the presence of GTP, this assembly is transient as the coat binds and falls apart. There was no difference in the recruitment or release of the coat when reactions containing wild-type Sec24p and Sec24p-m11 were compared. Download figure Download PowerPoint We tested the stability of the coat generated with Sec24p-m11 using liposome binding studies that permit the hierarchical assembly of the coat on synthetic liposomes (Matsuoka et al, 1998). Sar1p, Sec23/24p and Sec13/31p were incubated with liposomes in the presence of GDP, GTP or GMP-PNP (Figure 3C). As expected, the coat failed to assemble in the presence of GDP, whereas GMP-PNP permitted binding of each component to the liposome, regardless of the form of Sec24p included. Similarly, the GTP-containing incubation yielded relatively poor binding of the whole coat, consistent with rapid GTP hydrolysis and coat turnover during the course of the incubation. The presence of the Sec24p-m11 mutant had no stabilizing effect and showed the same limited coat assembly as the wild-type incubation. We confirmed that the Sec24p-m11 mutant did not support stable coat assembly using a light-scattering assay that monitors coat binding and release in real time (Figure 3D), which showed wild-type and mutant Sec24p with similar kinetics of coat assembly and disassembly. Consistent with these findings, the Sec24p-m11 mutant did not have any effect on the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p when monitored either by tryptophan fluorescence (EAM, unpublished observations) or by a 33P-GTP hydrolysis assay (see Figure 5). Our combined biochemical analyses did not allow us to determine the molecular function of the m11 site, so we undertook a genetic approach to gain better insight into the cellular pathways that are influenced by this mutation in vivo. Genetic analysis of the sec24-m11 mutant by synthetic dosage lethality screening We used an approach known as synthetic dosage lethality (SDL; Kroll et al, 1996) to search for genetic backgrounds in which overexpression of Sec24p-m11 is toxic. We placed SEC24 and sec24-m11 under the control of a Cu2+-inducible promoter, and systematically introduced these plasmids into the haploid deletion collection (Reid et al, 2011). Expression of SEC24 or sec24-m11 was induced by transfer to Cu2+-containing medium and strain growth was scored using ScreenMill software (Dittmar et al, 2010). Strains exhibiting slow growth (P-value significance <0.02) when expressing an empty plasmid were discarded as likely off-target mutants that cannot tolerate the high concentrations of Cu2+ required to induce expression. The remaining SDL hits were further filtered to exclude strains that were affected by overexpression of both SEC24 and sec24-m11. The final analysis yielded 131 strains that were specifically impaired in their ability to tolerate overexpression of the mutant protein (Supplementary Table S1). Of these, a large proportion (50 in total) were known genes involved in ER or Golgi function, or were uncharacterized components that reside in the ER or Golgi. We retested the overexpression lethality associated with the m11 mutant in these strain backgrounds and confirmed 40 validated hits (Supplementary Figure S2). The majority of m11-specific hits function in various aspects of Golgi vesicle formation or fusion, and the bulk of these mutants have previously been defined as having negative genetic interactions with a hypomorphic allele of sec24 (Schuldiner et al, 2005). Of particular interest to our focus on vesicle formation in the ER, was sed4, which had not been previously identified as showing a genetic interaction with SEC24, although genetic interactions with other early secretory components such as SAR1 and SEC16 have been defined (Gimeno et al, 1995; Saito et al, 1999). Sed4p is an integral ER membrane protein with homology to the Sar1p guanine nucleotide exchange factor (GEF), Sec12p, but lacking any GEF activity (Saito-Nakano and Nakano, 2000), and with poorly defined function (Saito-Nakano and Nakano, 2000; Kodera et al., 2011). We confirmed the specificity of the sec24-m11 SDL with sed4Δ by placing SEC24 under the control of the GAL1 promoter and testing the effect of the m11, A-, B- or C-site mutations. Wild-type cells tolerated overexpression of all forms of SEC24, whereas the sed4Δ strain was specifically sensitive to expression of the sec24-m11 allele (Figure 4A; LK, unpublished data). Similarly, we tested true synthetic lethality of a sed4Δ null mutation combined with various alleles of SEC24. Only the sec24-m11 mutant was unable to grow in a sed4Δ strain whereas cargo-binding alleles of SEC24 were viable in this background (Figure 4B). These genetic interactions suggest that cells expressing the Sec24p-m11 mutant are impaired in a cellular process that also involves Sed4p; in the absence of normal Sec24p activity, a functional copy of Sed4p is required to maintain viability. The molecular function of Sed4p is not understood and appears to be specific for S. cerevisiae and closely related species since no obvious orthologue has been identified in more distant species. Overexpression of SED4 suppresses the lethality associated with mutations in either SEC16 or SAR1 (Gimeno et al, 1995; Saito et al, 1999). These genetic interactions coupled with the fact that the cytoplasmic domain of Sed4p binds to the C-terminal domain of Sec16p suggests that it acts in conjunction with Sec16p to modulate the function of Sar1p (Gimeno et al, 1995; Saito et al, 1999; Saito-Nakano and Nakano, 2000; Kodera et al., 2011). Orthologues of Sec16p have been characterized in higher eukaryotes and their role in COPII vesicle formation is well established (Watson et al, 2006; Bhattacharyya and Glick, 2007; Iinuma et al, 2007; Hughes et al, 2009), although the exact molecular function remains unknown, which led us to test whether the sec24-m11 mutant showed similar genetic interactions with a sec16 mutant. Sec16p is an essential protein and is therefore not represented in the haploid deletion collection used in our SDL screen. Therefore, we tested a temperature-sensitive mutant of sec16, which carries a Leu-Pro substitution in residue 1088 (Espenshade et al, 1995) for a genetic interaction with SEC24. When Sec24p-m11 was the sole copy of Sec24p in a sec16-2 background, cells were unable to grow at the normally permissive temperature of 25°C (Figure 4B). This synthetic lethality was not observed with the canonical cargo-binding mutants of Sec24p (Figure 4B), suggesting a unique feature of the m11 mutant is a perturbation in Sed4p/Sec16p-mediated regulation of vesicle budding. Figure 4.Expression of Sec24p-m11 is toxic in sed4Δ and sec16-2 strains. (A) Wild-type SEC24 or sec24-m11 was placed under the control of the inducible GAL1 promoter and introduced into either wild-type (BY4742, upper panel) or sed4Δ strains (lower panel). Serial dilutions of cells were plated onto media containing galactos
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