1540 Research Article Multicopy suppressor analysis of thermosensitive YIP1 alleles implicates GOT1 in transport from the ER

Andrés Lorente-Rodríguez, Matthew Heidtman* and Charles Barlowe‡ Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA *Present address: Department of Microbiology, Tufts University, Boston, MA 02111, USA ‡Author for correspondence (e-mail: [email protected])

Accepted 29 January 2009 Journal of Cell Science 122, 1540-1550 Published by The Company of Biologists 2009 doi:10.1242/jcs.042457

Summary Yip1p belongs to a conserved family of membrane-spanning SEC31 , which encode subunits of the COPII coat. Further proteins that are involved in intracellular trafficking. Studies characterization of Got1p revealed that this protein was have shown that Yip1p forms a heteromeric integral membrane efficiently packaged into COPII vesicles and cycled rapidly complex, is required for biogenesis of ER-derived COPII between the ER and Golgi compartments. Based on the findings vesicles, and can interact with Rab GTPases. However, the role we propose that Got1p has an unexpected role in vesicle of the Yip1 complex in vesicle budding is not well understood. formation from the ER by influencing membrane properties. To gain further insight, we isolated multicopy suppressors of the thermosensitive yip1-2 allele. This screen identified GOT1, Supplementary material available online at FYV8 and TSC3 as novel high-copy suppressors. The strongest http://jcs.biologists.org/cgi/content/full/122/10/1540/DC1 suppressor, GOT1, also displayed moderate suppressor activity toward temperature-sensitive mutations in the SEC23 and Key words: ER, Golgi, COPII vesicles, Trafficking, Yip1 proteins

Introduction one another and a larger family of polytopic membrane proteins, Intracellular transport between membrane-bound compartments of known as the Yip1 family (Calero et al., 2002). Our studies have the early secretory pathway is highly dynamic, yet organellar shown that the Yip1 complex acts in a later stage of vesicle budding, identity is strictly maintained. Newly made secretory proteins and most likely after assembly of the COPII coat. We propose the Yip1 lipids are selected for anterograde transport from the endoplasmic complex regulates COPII budding at the membrane scission stage reticulum (ER) to the Golgi complex while compartmental residents (Heidtman et al., 2005). Yip1 family members might act similarly are retained and/or very efficiently retrieved. Organization of the in other coat-dependent budding events at distinct intracellular

Journal of Cell Science early secretory pathway is known to depend on coat protein compartments. To gain further insight on Yip1 complex function, complexes, membrane targeting and fusion factors and a host of we isolated genetic suppressors of the semi-functional yip1-2 additional components that catalyze bidirectional transport between mutation. Three multicopy suppressor genes were identified, all of the ER and Golgi (Bonifacino and Glick, 2004). However, the which might in some manner be connected to membrane structure overall mechanisms that govern transport and maintain organelle and function in the early secretory pathway. The strongest suppressor structure and function in the early secretory pathway are poorly isolated encodes Got1p, a 16 kDa tetraspanning membrane protein understood. implicated in Golgi transport. Based on further investigation in this The current view of ER export suggests that folded secretory study we propose that Got1p influences trafficking from the ER. proteins are collected at specialized zones in the ER, referred to as ER-exit sites. The COPII-budding machinery is also concentrated Results at ER-exit sites where cargo proteins are linked directly or indirectly Identification of GOT1, FYV8 and TSC3 as novel high-copy to coat subunits for their incorporation into COPII-derived transport suppressors of the yip1-2 strain intermediates (Lee et al., 2004). The levels of specific secretory Recent biochemical, genetic and morphological studies have shown cargo and lipid species appear to influence ER export dynamics that the Yip1p complex has a crucial role in COPII vesicle (Forster et al., 2006; Runz et al., 2006). However, it is not clear biogenesis (Heidtman et al., 2003; Heidtman et al., 2005). To how vesicle budding is regulated at ER-exit sites under a variety identify other products that function in the same pathway as of these conditions. We have identified additional protein this complex, we performed a multicopy suppression screen of the components on COPII transport vesicles that cycle between ER and themosensitive yip1-2 strain (Yang et al., 1998). This strain contains Golgi compartments and influence ER export (Otte et al., 2001). a single point mutation (G175E) in the second predicted One such protein, Yip1p, is efficiently packaged into vesicles and transmembrane domain of Yip1p, which results in growth defects required for COPII budding (Heidtman et al., 2003). and an accumulation of secretory proteins in the ER at restrictive Yip1p was initially identified as a Ypt/Rab-interacting protein temperatures (Yang et al., 1998). A high-copy genomic YEp24 and localizes to ER and Golgi membranes (Yang et al., 1998). Yip1p library bearing the URA3 marker (Carlson and Botstein, 1982) was forms a heteromeric integral membrane complex with two other used to select for overexpressed genes that could rescue the yip1-2 membrane proteins, Yif1p and Yos1p (Matern et al., 2000; Heidtman growth defect at a restrictive temperature of 36°C. From a pool of et al., 2005). All three members of the Yip1 complex are conserved ~10,000 transformants, we identified 13 plasmids that suppressed in nature and both Yip1p and Yif1p share sequence identity with the yip1-2 mutation upon retransformation. DNA sequencing GOT1 in ER-Golgi transport 1541

identified the genomic insert present in each of the suppressing plasmids. Ten of these plasmids contained a region of DNA harboring the YIP1 open reading frame (ORF). The remaining three plasmids contained regions of DNA corresponding to II, VII and XIII respectively. The suppressing plasmid bearing a region of VII had only one complete ORF present: FYV8 (function required for yeast viability upon toxin exposure). FYV8 is a non-essential gene; however, strains lacking FYV8 exhibit hypersensitivity to K1 killer toxin (Page et al., 2003). This gene is predicted to encode a ~90 kDa protein with moderate homology to predicted open reading frames in higher eukaryotes and might be involved in the response to ER stress (Chen et al., 2005). To more closely examine the level of FYV8 high-copy suppression activity, we performed dilution series experiments to monitor growth rates. As shown in Fig. 1A, FYV8 displayed mild but detectable suppression of the yip1-2 strain at 36°C. Multicopy FYV8 did not suppress the thermosensitive growth defects of other yip1 or yif1 conditional mutants at the restrictive temperature (data not shown). The suppression activity of FYV8 towards the yip1-2 strain was not investigated further. The suppressing plasmid containing a region of chromosome II also had only a single complete ORF present: TSC3 (temperature- sensitive suppressor of Ca2+ sensitivity). The TSC3 gene encodes a ~9 kDa transmembrane protein that localizes to ER membranes. Several lines of evidence indicate that Tsc3p participates in the early steps of sphingolipid synthesis at the ER (Gable et al., 2000). As shown in Fig. 1A, TSC3 moderately suppressed the growth defect of the yip1-2 strain at 36°C. Multicopy TSC3 did not suppress the thermosensitive growth defects of other yip1 or yif1 conditional mutants (data not shown). The mechanism by which multicopy TSC3 suppressed the yip1-2 mutation was not investigated. The third suppressor plasmid isolated from this screen contained a region of DNA from chromosome XIII. Several complete ORFs were present in this region, including YMR291W, GOT1, YMR293C

Journal of Cell Science and JNM1. Subcloning revealed that GOT1 was solely responsible for the suppression of the thermosensitive growth defect of the yip1-2 strain (Fig. 1B). GOT1 (Golgi transport) is a non-essential Fig. 1. Multicopy suppressors of the yip1-2 strain. Transformed wild-type gene encoding a tetra-spanning integral membrane protein of (MSUC-3D) and yip1-2 (YXY12α) strains were grown to saturation in YMD- ~16 kDa and was originally identified in a synthetic-lethal screen URA to maintain selection of plasmids, adjusted to an OD(600) of 1.0, and designed to isolate mutants that require the SFT2 gene for function 5 μl of tenfold dilutions were spotted onto YPD plates. Plates were incubated at 25°C or 36°C for 48 hours. (A) Comparison of multicopy suppression of (Conchon et al., 1999). Both Got1p and Sft2p are tetra-spanning yip1-2 by FYV8 and TSC3. (B) Comparison of multicopy suppression of yip1-2 membrane proteins that are conserved across species. Double- by GOT1. The dilution series experiments in A and B were conducted on mutant strains that rely on conditional got1-2 or sft2-2 alleles for separate occasions. growth exhibit secretion defects in pulse-chase assays and accumulate ER membranes indicating a role for these proteins in the early secretory pathway (Conchon et al., 1999). As shown in of SFT2 displayed suppressor activity toward the yip1 or yif1 Fig. 1B, we found that multicopy GOT1 strongly suppressed the conditional alleles. We observed that multicopy SFT2 was unable growth defect of the yip1-2 strain at 36°C to the same extent as to suppress the growth defects associated with any of the yip1 or multicopy YIP1 or YIF1. Furthermore, multicopy GOT1 exhibited yif1 mutant strains tested (supplementary material Fig. S1). These different degrees of suppression toward the yip1-1 (P114, G129E), results suggest that the strong suppressor activity of GOT1 toward yip1-4 (E70K), yif1-2 (W133Amber) and yif1-4 (V162A, S189P, these yip1 and yif1 alleles was relatively specific. L205A, V230A) mutants at their restrictive temperatures (Fig. 2; and data not shown). We also tested a conditional allele in YOS1, High-copy GOT1 suppresses other early secretory pathway which encodes an additional subunit of the Yip1-Yif1 complex thermosensitive mutants (Heidtman et al., 2005), and observed that multicopy GOT1 did not In order to determine if multicopy GOT1 is a specific suppressor suppress the growth defect of a yos1-1 (N18A) strain (Table 1). of Yip1 complex mutants, or more generally could suppress other Got1p and Sft2p are proposed to have partially redundant roles ER-Golgi trafficking mutants, we transformed several in intracellular trafficking with Got1p acting in the early secretory thermosensitive strains with multicopy GOT1 or an empty vector pathway and Sft2p functioning at the late Golgi (Conchon et al., control. For each mutant analyzed, two independent transformants 1999). Although multicopy SFT2 was not isolated in our suppressor were grown to saturation in the appropriate selective media to screen, we wanted to test directly whether a functional 2 μm version maintain plasmids, then tenfold dilution series were spotted onto 1542 Journal of Cell Science 122 (10)

Table 1. Summary of multicopy suppression interactions* 2 μ GOT1 suppression Process Strain 34°C 38°C Budding sec12-4 –– sec16-2 –– sec13-1 –– sec31-1 ++ sec23-1 +– sec24-11 ±– sec24-13 –– yif1-2 +† ND yif1-4 ++† ND yip1-1 ++† ND yip1-2 +++† ND yip1-4 ++ + yos1-1 –– Tethering ypt1-1 –– ypt1-3 –– uso1-1 ±± sec34-2 (cog3) –– sec35-1 (cog2) –– Fusion sly1-TS –– sec17-1 –– sed5-1 –– bos1-1 –– bet1-1 ±– sec22Δ –– Retrograde transport ret1-1 –– sec21-1 ––

*Symbols indicate: +++, wild-type; ++, near wild-type; +, intermediate; ±, poor; –, no growth; ND, not determined; blank, mutant grows as wild-type at indicated temperature. †Growth tested at 36°C.

prepared polyclonal antibodies specific for Got1p to detect the endogenous untagged protein. For localization studies, cell lysates were prepared from wild-type and got1Δ strains to monitor the

Journal of Cell Science subcellular distribution of Got1p on sucrose gradients. Fractions were collected and analyzed for Got1p, the early Golgi marker Och1p, the ER marker Sec61p, and the COPII vesicle protein Yip1p. Fig. 2. GOT1 is a multicopy suppressor of the yip1-1 and yip1-4 strains. The experiment was carried out as in Fig. 1. (A) Multicopy GOT1 suppression of We observed that ~66% of Got1p cosedimented with Sec61p and yip1-1 (YXY11a) compared with the wild type (MSUC-3D). (B) The yip1-4 ~34% with Och1p (Fig. 3A). This fractionation pattern was similar strain (RCY1764) compared with the wild type (RCY1768) at restrictive to that of Yip1p and other vesicle proteins that cycle between the temperatures. ER and Golgi compartments (Cao and Barlowe, 2000; Heidtman et al., 2005; Otte et al., 2001; Powers and Barlowe, 1998; Schimmoller et al., 1995). No differences were observed when the YPD plates and growth monitored at 30°C, 34°C and 38°C. In sucrose gradient fractionation patterns for Yip1p, Sec61p and addition to yip1-4 and yos1-1 mutants, we tested a total of 20 mutant Och1p were compared in wild type and got1Δ strains (data not strains with deficiencies in the stages of ER vesicle budding, vesicle shown), indicating that GOT1 is not required for Yip1p localization. tethering, membrane fusion or retrograde transport. Interestingly, To confirm dynamic cycling of Got1p in vivo, we analyzed the multicopy GOT1 moderately suppressed the growth defects of distribution of Got1p after a sec12-4 block. Proteins that cycle sec31-1 and sec23-1 genes involved in vesicle budding, and between the ER and Golgi compartments accumulate in the ER weakly suppressed the growth defects of sec24-11, uso1-1 and when export from this compartment is blocked by shifting a sec12-4 bet1-1 (Table 1; supplementary material Fig. S2). strain to the restrictive temperature (Schroder et al., 1995). Wild- type and sec12-4 cells in logarithmic-phase growth were shifted to Got1p cycles between the ER and Golgi compartments 37°C for 45 minutes and membrane organelles resolved by Conchon and colleagues (Conchon et al., 1999) localized a C- differential centrifugation of cell lysates. The P13 fraction (enriched terminally tagged 3MYC version of Got1p to cis-Golgi membranes in ER membranes) and the P100 fraction (enriched in Golgi by fluorescence microscopy and subcellular fractionation membranes) were assessed by immunoblot (Fig. 3B). We observed approaches. Interestingly, a more recent study localized a C- that the distribution of Och1p and Sec61p was not affected by the terminally GFP-tagged version of Got1p to the ER (Huh et al., sec12-4 block. However, Got1p and the known COPII vesicle 2003). These results, although conflicting, could be consistent with proteins Erv41p and Erv25p, accumulated in the ER fraction of our genetic data, and suggested that Got1p might cycle between sec12-4 cells. In addition, a got1Δ strain and isogenic wild-type the ER and Golgi compartments. To investigate this hypothesis, we were examined under the temperature shift and no alterations in GOT1 in ER-Golgi transport 1543

Fig. 4. Got1p is efficiently packaged into COPII vesicles. Large scale budding reactions were performed on wild-type (CBY740) and got1Δ (CBY1574) microsomes as described in the Materials and Methods. Total membranes (T), budded vesicles from minus (–) and plus (+) COPII incubations were immunoblotted for Sec12p (ER marker), Erv41p and Erv25p (ER-Golgi cycling markers) and Got1p. The total lane represents 25% of a budding reaction. Note that the packaging of proteins into COPII vesicles was not affected in got1Δ membranes.

levels of individual proteins were quantified by densitometry analysis of immunoblots using ImageJ (Rasband, 1997-2005), and packaging efficiencies were calculated. As shown in Fig. 4, the ER- resident Sec12p was not efficiently packaged (1%) into COPII vesicles, compared with efficient packaging of Erv41p (37%) and Erv25p (25%). Notably, we found that Got1p was also very efficiently packaged (62%) into COPII vesicles. This level of packaging indicates that Got1p probably contains a sorting signal recognized by the COPII coat or might associate with other factors that contain potent ER-export signals. To determine whether the got1Δ mutation influences the sorting efficiency of other COPII vesicle proteins, the composition of vesicles produced from wild-type and got1Δ microsomes were compared by immunoblot. As seen in Fig. 4, the packaging Δ Journal of Cell Science efficiencies of Erv41p and Erv25p were not influenced by got1 mutation. In addition, no changes were detected in the packaging Fig. 3. Got1p cycles between the ER and Golgi compartments. (A) Got1p co- of other characterized ER vesicle proteins including Yip1p, Yif1p, fractionates with ER and Golgi markers. A whole-cell lysate from wild-type Erv26p, Sed5p, Bet1p, Bos1p and Sec22p (data not shown). cells (CBY740) separated on an 18-60% sucrose density gradient, and Moreover, ER-resident proteins such as Sec12p (Fig. 4), Sec61p fractions collected from the top. The top panel shows the fractionation pattern and Kar2p (not shown) were not inappropriately packaged into of Sec61p (ER marker), Och1p (Golgi marker) and Yip1p (ER-Golgi cycling Δ marker). The bottom panel shows the fractionation pattern of Got1p and Yip1p COPII vesicles when microsomes were prepared from got1 cells. with % sucrose based on refractometry readings indicated on the right axis. These data suggest that Got1p is not required for protein sorting at (B) Blocking ER export in the sec12-4 strain (37°C for 45 minutes) causes the ER, at least for the subset of proteins we examined. Got1p to accumulate in the ER fraction (P13). Erv25p and Erv41p, proteins that cycle between the ER and Golgi, also shift to the ER fraction. The first four lanes compare the wild type (RSY248) with sec12-4 (RSY263) and the In vitro analysis of Got1p function in ER-Golgi transport assays last four lanes compare wild type (CBY740) with got1Δ (CBY1574). We observed that multicopy GOT1 could not bypass a complete yip1Δ deletion (supplementary material Fig. S3), indicating that Got1p does not replace Yip1p function but somehow compensates protein distribution were observed. Taken together, these data for semi-functional yip1 and yif1 alleles. To explore this indicate that Got1p cycles between the ER and Golgi compartments compensation mechanism, we tested whether elevated levels of and that got1Δ does not influence the normal localization pattern Got1p could rescue the in vitro budding defect observed in of the set of proteins monitored. membranes prepared from yip1-4 mutant cells. Semi-intact cell membranes prepared from wild-type and yip1-4 cells bearing Got1p is efficiently packaged into COPII vesicles empty vector or multicopy GOT1 were used in a cell free assay Our localization data and genetic interaction analysis suggested that that measures COPII dependent vesicle budding of [35S]glyco-pro- Got1p would be packaged into COPII vesicles. To test this idea, α-factor (gpαf). As shown in Fig. 5A, mutant yip1-4 membranes microsomes from wild-type and got1Δ strains were incubated in displayed a significant budding defect compared with wild-type the presence (+) or absence (–) of COPII proteins with an energy- membranes, which is consistent with our previous results (Heidtman regeneration system to reconstitute budding. Membrane vesicles et al., 2003). Interestingly, we observed that multicopy GOT1 generated in each condition were isolated and analyzed by partially rescued the budding defect of yip1-4 membranes, similarly immunoblot as described (Belden and Barlowe, 1996). Relative to that observed for multicopy YOS1 rescue of the yip1-4 budding 1544 Journal of Cell Science 122 (10)

with Golgi membranes (Conchon et al., 1999). However, our investigation of the got1Δ strain from the Research Genetics strain collection (Winzeler et al., 1999) revealed that membranes prepared from this strain did not display detectable defects in any specific transport stages or in overall ER-Golgi transport (Fig. 5B,C). Upon re-analysis of the original got1Δ strain (Conchon et al., 1999), we did observe phenotypic consequences; however, these defects could not be restored by reintroduction of the GOT1 gene. Closer examination of membranes isolated from this original got1Δ strain also revealed that the Rab-GTPases Ypt1p and Ypt7p were upregulated and predominantly found in the soluble fraction instead of associated with membranes; again, this defect was not rescued by transformation with multicopy GOT1 (supplementary material Fig. S4). These results suggest that the original got1Δ strain contains additional mutation(s) or a genetic background that contributes to the observed trafficking defects. Further studies will be required to determine the nature of these genetic differences.

Got1p forms a low molecular weight homo-oligomeric complex We next took a biochemical approach to determine whether other proteins could be detected in stable association with Got1p. Our genetic analyses suggested that Got1p might physically associate with the Yip1 complex or other proteins involved in vesicle budding. To investigate this, we prepared microsomes from a strain expressing Yif1p-3HA as the sole source of cellular Yif1p (Heidtman et al., 2005). Microsomes from this strain were solubilized with 0.5% digitonin and Yif1p-3HA immunoprecipitated with monoclonal anti-HA. As seen in Fig. 6A, Yif1p-3HA was efficiently recovered, and Yip1p and Yos1p – both members of the Yip1 complex – were specifically co-immunoprecipitated (Heidtman et al., 2005; Matern et al., 2000). When the same immunoprecipitates were examined for the presence of Got1p using polyclonal anti- Got1p antibodies, no Got1p was detected. Based on this result we conclude that Got1p is not a stable member of the Yip1 complex.

Journal of Cell Science In a reciprocal approach, we immunoprecipitated Got1p to identify potential binding partners. Using a CEN plasmid expressing Got1p- Fig. 5. In vitro analysis of Got1p function in ER-Golgi transport assays. 3MYC from the TPI promoter (Conchon et al., 1999), we transformed (A) Multicopy GOT1 partially restores COPII budding from yip1-4 the Yif1p-3HA strain to express a tagged version of Got1p. membranes. Washed semi-intact cell membranes containing [35S]gpαf were Microsomes were prepared from these strains and then solubilized prepared from wild-type (RCY1768 with pRS426), yip1-4 (RCY1764 with with 1% Triton X-100. The solubilized material was subjected to pRS426) and yip1-4/GOT1 (RCY1764 with pRS426-GOT1) strains. Membranes were mock treated (NA) or incubated with COPII proteins at immunoprecipitation with monoclonal anti-Myc antibodies. As 29°C for 20 minutes and [35S]gpαf packaged into budded vesicles measured shown in Fig. 6B, Got1p-3MYC was efficiently recovered in this as described in the Materials and Methods. (B) Membranes lacking Got1p do manner as determined by immunoblotting with anti-Myc and not display budding or tethering defects in vitro. Washed semi-intact cell polyclonal anti-Got1p antibodies. Interestingly, we found that membranes as above were prepared from wild-type (CBY740) and got1Δ (CBY1574) strains. Membranes were mock treated (NA), incubated with endogenous Got1p was efficiently co-immunoprecipitated with COPII proteins or COPII proteins plus Uso1p at 23°C for 30 minutes and Got1p-3MYC, indicative of a homo-oligomeric association. Yif1p- freely diffusible vesicles quantified to assess levels of vesicle budding and 3HA, Sec23p and Sec61p did not co-immunoprecipitate with Got1p- tethering. (C) Membranes lacking Got1p do not display in vitro transport 3MYC, confirming the specificity of the Got1p interaction. Further defects. Washed semi-intact cell membranes as above were mock treated analysis of these immunoprecipitates failed to detect Sec12p, Sec13p, (NA) or incubated with COPII, Uso1p and LMA1 proteins to reconstitute (Recon) transport. After 70 minutes at 23°C, the amount of Golgi-modified Sec24p, Yip1p, Erv41p, Uso1p, Sec22p, Bet1p, Bos1p or Sed5p in [35S]gpαf was measured to determine transport efficiency. Error bars association with Got1p-3MYC (data not shown). Moreover, analysis represent the range of duplicate determinations. of Got1p-3MYC immunoprecipitates on stained protein gels did not reveal other binding partners beyond endogenous Got1p. Similar results were obtained when immunoprecipitation experiments were defect (Heidtman et al., 2005). Taken together, these results indicate performed in 0.5% digitonin in place of 1% Triton X-100 (data not that elevated levels of Got1p can partially compensate for reduced shown). Yip1p activity in this COPII vesicle budding assay. To further characterize the nature of the Got1p oligomer, we We next tested the influence of got1Δ deletion mutation on other determined its relative size by sucrose gradient sedimentation analysis. stages in ER-Golgi transport. Previous studies indicated that A post-nuclear fraction of homogenized cells was solubilized in 1% membranes lacking Got1p budded COPII vesicles containing Triton X-100 and proteins resolved on a linear 1-12% sucrose velocity [35S]gpαf at normal levels but were compromised for vesicle fusion gradient containing 1% Triton X-100. Soluble globular molecular GOT1 in ER-Golgi transport 1545

weight markers (RNAseA: 13.7 kDa; Aldolase: 158 kDa; and Catalase: 232 kDa) were run in parallel gradients. Erv25p was used as a control for the resolution of integral membrane protein complexes because Erv25p is known to form a ~100 kDa complex with Emp24p, Erp1p and Erp2p (Marzioch et al., 1999). Immunoblot analysis of sucrose gradients (Fig. 6C) showed that Got1p peaked between fractions 2 and 3 at a size slightly greater than Sec22p (~25 kDa). Taken together, our results suggest that Got1p most likely forms a low molecular weight homo-dimeric complex.

GAL1 overexpression of Got1p causes a block in ER-Golgi transport We have shown that ~tenfold overexpression of GOT1 from a 2 μm plasmid (supplementary material Fig. S4) suppresses thermosensitive mutations in essential genes required for COPII vesicle budding (Table 1). To investigate dosage effects of GOT1 expression, we placed GOT1 under control of the inducible GAL1 promoter. In the case of GAL1-3HA-GOT1 expression, we Journal of Cell Science

Fig. 6. Immunoprecipitation analysis of Got1p. (A) Got1p was not recovered with the Yip1 complex. Digitonin solubilized microsomes from wild type (FY834) or Yif1p-3HA (CBY801) strains were mock precipitated (–Ab) or precipitated with anti-HA antibody (+Ab). Total (T) and solubilized (S) samples represent 1.5% of the starting material. Note specific co- immunoprecipitation of Yip1p and Yos1p with Yif1p-3HA. (B and C) Got1p forms a low molecular weight homo-oligomer. (B) Triton-X-100-solubilized microsomes from Yif1p-3HA (CBY801) or Yif1p-3HA/Got1-3MYC Fig. 7. GAL1 overexpression of 3HA-Got1p inhibits growth and causes a (CBY1373) strains were mock precipitated (–Ab) or precipitated with anti- secretory block. Wild-type (CBY740) and GAL1-3HA-GOT1 (CBY2577) cMyc antibody (+Ab). Total (T) and solubilized (S) samples represent 1.0% of strains were pre-cultured in medium with 1% galactose and 1% glucose and the starting material, whereas the unbound (U) lane represents 1.7% of the then shifted to medium with 2% galactose. (A) Cultures were analyzed for total unbound material. Note that the strain expressing Got1p-3MYC also growth over time by absorbance. (B) At the 5 hour time-point, cell extracts expresses endogenous Got1p. The arrow indicates Got1p-3MYC, the asterisk were prepared and proteins monitored by immunoblot. Arrowheads identify indicates position of anti-Myc antibody light chain and the arrowhead the position of 3HA-Got1p and arrows indicate untagged Got1p. The panel indicates a crossreacting species. (C) Detergent-solubilized membrane proteins labeled ‘(dark)’ shows a saturated exposure of the anti-Got1p blot to visualize were resolved on a 1-12% sucrose velocity gradient containing 1% Triton X- endogenous levels of protein. The ER (p1), Golgi (p2) and mature (m) forms 100. Globular molecular mass markers were run on parallel gradients (arrows of CPY are indicated. Note the reduced growth rate and accumulation of the above graphs); Erv25 complex (~100 kDa) and Sec22p were internal controls. ER form of CPY in the GAL1-3HA-GOT1 strain. 1546 Journal of Cell Science 122 (10)

To inspect organelle morphology, we monitored the ER marker Sec63-GFP (Prinz et al., 2000), and the late-Golgi marker Sec7- GFP (Rossanese et al., 2001) by microscopy after growth in 2% galactose for 5 hours (Fig. 8). ER morphology was assessed by focusing on the center of cells, where wild-type cells exhibited a typical perinuclear and peripheral ER morphology (Fig. 8A, WT) (Prinz et al., 2000). GAL1 overexpression of HA-Got1p produced an elaboration of ER membranes, which was accompanied in a few cases by gapped ER structures (Fig. 8A, GAL1-3HA-GOT1). After assessing ER structures in a population of cells under each condition, we determined that 60% of the GAL1-3HA-GOT1 cells displayed abnormal ER morphologies whereas only 20% of the control cells contained such structures (supplementary material Fig. S5A). Previous reports have shown that yeast mutants blocked in ER export, such as sec23-1 (Prinz et al., 2000) and yip1-4 (Heidtman et al., 2003), accumulate similar ER structures at restrictive temperatures. Golgi membranes were assessed by monitoring the late-Golgi marker Sec7-GFP, which localizes to punctate structures throughout wild-type cells and displays minimal background GFP staining (Fig. 8B: WT) (Seron et al., 1998; Rossanese et al., 2001). By contrast, GAL1-3HA-GOT1 cells exhibited a diffuse Sec7-GFP localization pattern with punctate structures reduced or absent from cells (Fig. 8B: GAL1-3HA-GOT1). Quantification of these phenotypes indicated that 95% of the GAL1-3HA-GOT1 cells displayed abnormal Sec7-GFP distributions compared with 20% of cells in the wild-type culture (supplementary material Fig. S5B). Similar results were obtained from cells expressing the early-Golgi marker Sec21p-GFP (data not shown). Loss of early- and late-Golgi structures has been reported in yeast mutants under conditions that block ER export as well as in mutants that block vesicle fusion stages with Golgi membranes (Wooding and Pelham, 1998; Sato and Nakano, 2002; Kamena et al., 2008). This dispersal of Golgi Fig. 8. GAL1 overexpression of 3HA-Got1p causes abnormal ER and Golgi membranes is thought to be caused by a reduced delivery of ER morphologies. (A) Wild-type and GAL1-3HA-GOT1 strains expressing Sec63- Journal of Cell Science derived cargo to the Golgi. In summary, our observations that GAL1- GFP (CBY2537 and CBY2542, respectively) were cultured in 2% galactose as 3HA-GOT1 alters ER and Golgi morphologies, and causes an in Fig. 7 and observed by fluorescence and bright field microscopy at the 5 hour time point. Note the elaboration of ER membranes and appearance of accumulation of the ER form of CPY, indicate high levels of 3HA- membrane gaps in the GAL1-3HA-GOT1 strain. (B) Wild-type and GAL1- Got1p inhibit transport through the early secretory pathway. These 3HA-GOT1 strains expressing Sec7-GFP (CBY2538 and CBY2543, results are consistent with a block at the ER-export stage. respectively) were treated as described in A. Note that GAL1-3HA-GOT1 cells Alternatively, these high levels of HA-Got1p could influence display a diffuse Sec7-GFP localization pattern with punctate Golgi structures reduced or absent. Scale bars: 5 μm. A quantitative analysis of these abnormal subsequent stages in transport to the Golgi complex. ER and Golgi morphologies in a population of cells is provided in supplementary material Fig. S5. Discussion Previous studies implicated Yip1p and Yif1p in ER-Golgi trafficking (Yang et al., 1998; Matern et al., 2000) and more recent work has observed that increases in galactose concentration in growth demonstrated a requirement for the Yip1 complex in COPII vesicle medium significantly reduced growth rates compared with wild- budding (Heidtman et al., 2003; Heidtman et al., 2005). However, type strains. Indeed, strains containing GAL1-3HA-GOT1 did not the molecular mechanism by which the Yip1 complex contributes grow on medium with 2% galactose. To explore the cause of this to this process is unclear. To gain a better understanding of the role toxicity, we precultured cells in 1% glucose, 1% galactose and of this complex in COPII vesicle budding, we performed a then shifted cultures to 2% galactose. After 1.5 hours, a reduction multicopy suppressor screen of the thermosensitive yip1-2 strain, in growth rate was detected and after 5 hours, growth on galactose which contains a single point mutation (G175E) in the second medium was strongly inhibited (Fig. 7A). At the 5 hour time-point, predicted transmembrane domain of Yip1p (Yang et al., 1998). Three we monitored secretory protein transport and organelle multicopy suppressors of yip1-2 were identified (FYV8, TSC3 and morphology. As seen in Fig. 7B, the ER form of carboxypeptidase GOT1) and the properties of GOT1 were further characterized. Y (p1 CPY) accumulated in the GAL1-regulated strain compared The FYV8 multicopy plasmid displayed modest suppressor with the wild type. Precursor accumulation correlated with an activity toward yip1-2. FYV8 was first identified in a screen for approximate 40-fold increase in 3HA-Got1p expression over deletion mutants with altered sensitivity to K1 killer toxin and fyv8Δ endogenous levels. This phenotype suggested that GAL1-3HA- cells are hypersensitive to K1 killer toxin, calcofluor white, GOT1 overexpression produces a block in ER-Golgi transport of hygromycin B, and SDS (Page et al., 2003). The cellular function secretory proteins. of Fyv8p remains unclear, although sequence-profiling methods GOT1 in ER-Golgi transport 1547

predict a function as a transcriptional regulator (Mi et al., 2005). also observed that multicopy GOT1 could not bypass a complete Recent studies suggest a role in the ER-stress response, because yip1Δ deletion, indicating that Got1p activity was not redundant fyv8Δ cells display increased sensitivity to tunicamycin and reducing with Yip1p. However, multicopy GOT1 was able to partially rescue agents (Chen et al., 2005). Other studies have implicated FYV8 in the budding defect of yip1-4 membranes suggesting that chromosome segregation (Baetz et al., 2004) and chromosome overexpression imparted membrane properties that compensate for stability (Smith et al., 2004). Based on the collective findings, we deficiencies in Yip1 complex activity. speculate that multicopy FYV8 upregulates genes involved in Consistent with previous reports on the localization of Got1p managing ER stress and this might allow yip1-4 mutant cells to (Conchon et al., 1999; Huh et al., 2003), we found that Got1p was cope with a deficit in Yip1p activity. very efficiently packaged into COPII vesicles and cycled between TSC3 was initially identified in a temperature-sensitive the ER and Golgi compartments. In contrast to previous findings, suppressor screen of Ca2+-sensitive csg2Δ mutants (Beeler et al., however, we observed that got1Δ membranes prepared from the 1998). Further investigation revealed that Tsc3p localizes to the ER Research Genetics isolate did not display defects in cell free assays where it associates with Lcb2p and is a subunit of the serine that monitored vesicle budding, tethering or fusion. Immunoblot palmitoyltransferase (SPT), which catalyzes the first committed step analysis of this strain confirmed that Got1p was not expressed. in sphingolipid synthesis (Gable et al., 2000; Monaghan et al., 2002). Moreover, addition of anti-Got1p immunoglobulins or anti-HA Tsc3p interaction with SPT stimulates activity several antibodies to GOT1-3HA membranes did not inhibit in vitro transport fold (Gable et al., 2000) by conferring substrate specificity to the even in a sft2Δ background (data not shown). These results suggested (Cowart and Hannun, 2007). Interestingly, a recent study that Got1p does not function in ER-to-Golgi transport as initially suggested a possible synthetic interaction between TSC3 and RUD3 proposed (Conchon et al., 1999). We found that the original got1Δ (Schuldiner et al., 2005), which might indicate a role for isolate appeared to contain mutation(s) that cause upregulation and sphingolipids in ER-Golgi trafficking. The identification of TSC3 mislocalization of the Rab GTPases Ypt1p and Ypt7p, which might as a dosage suppressor of the yip1-2 strain could be due to an explain the previously observed defect in fusion of COPII vesicles increased concentration of sphingolipids at the ER caused by high with Golgi membranes (Conchon et al., 1999). This defect was absent levels of Tsc3p. We have proposed that the Yip1 complex acts in from the Research Genetics got1Δ strain. Together, these results membrane dynamics through promotion of COPII vesicle scission indicate that Got1p influences transport though the early secretory (Heidtman et al., 2005). If increased sphingolipid levels at the ER pathway but under our in vitro conditions this function is not directly rescues impaired activity of the yip1-2 mutant, this would suggest required for a single round of ER-to-Golgi transport. that steps in the COPII-budding process are influenced by both A study in mammalian cells reported that overexpression of an N- membrane lipid composition and Yip1 complex activity. One such terminal GFP-tagged allele of the mammalian Got1p homolog causes possible role in budding might be to mediate membrane bending a delay in surface expression of VSV-G concomitant with defects in at the bud-neck to resolve membrane buds into vesicles. Golgi integrity (Starkuviene et al., 2004). In agreement with these GOT1 displayed the strongest multicopy suppressor activity findings, we observed that overexpression of 3HA-GOT1 from the toward yip1-2. GOT1 was originally identified in a synthetic lethal GAL1 promoter resulted in ER-to-Golgi transport defects screen with sft2Δ, a multicopy suppressor of thermosensitive alleles accompanied by growth arrest and altered ER and Golgi

Journal of Cell Science of sed5 (Banfield et al., 1995; Conchon et al., 1999). Both Got1p morphologies. These findings suggest that excessive levels of Got1p and Sft2p are small tetra-spanning membrane proteins that are may titrate out an essential component of the ER-to-Golgi transport conserved across species and have been proposed to have a role in machinery. Although no clear COPII- or COPI-packaging signals facilitating Sed5-dependent fusion events with the Golgi at early- were detected, Got1p was very efficiently packaged into COPII and late-Golgi compartments, respectively (Conchon et al., 1999). vesicles and it seems plausible that GAL1 overexpression of Got1p Furthermore, both Got1p and Sft2p have been proposed to act could saturate or somehow interfere with the normal cargo recognition downstream of trans-SNARE complex formation in promoting functions of the Sec23-Sec24 complex (Miller et al., 2003). membrane fusion pores (Bayer et al., 2003). However, we observed GOT1 has also been identified as a multicopy suppressor of the that the multicopy suppressor activity displayed by GOT1 was thermosensitive ypk1-1/ykr2Δ strain. Ypk1p and Ykr2p are distinct since multicopy SFT2 did not suppress temperature sensitive functionally redundant protein kinases proposed to function in the yip1 or yif1 mutants. These findings led us to further explore the maintenance of cell wall integrity (Roelants et al., 2002). role of Got1p in ER-to-Golgi transport and to gain a better Interestingly, phytosphingosine (the product of the third step in understanding of its relationship with the Yip1 complex. sphingolipid biosynthesis) is an activator of Pkh1p and Pkh2p Our findings were unexpected. We found that in addition to kinases, which in turn activate Ypk1 and Ykr2 kinases during the yip1-2, multicopy GOT1 was a strong suppressor of other heat stress response (Dickson et al., 2006; Roelants et al., 2002). thermosensitive alleles of yip1 and yif1, a moderate suppressor of Although Got1p localizes primarily to the early secretory pathway, sec31-1 and sec23-1, and weak suppressor of sec24-11, bet1-1 and we suggest that this rescue may be caused by an alteration in delivery uso1-1. This was a surprising finding in light of the fact that Got1p of plasma membrane lipids (possibly sphingolipids) involved in this was initially reported to act in membrane fusion (Conchon et al., signaling cascade. 1999). A relative specificity to the COPII-budding stage is supported These collective genetic findings seem to connect Got1p to a by the fact that thermosensitive mutations in 11 other genes function in regulation of membrane composition. We also note that involved in vesicle tethering, fusion and retrograde transport (COPI sequence-profiling programs indicate that Got1p contains a lipase- subunits) were not suppressed by multicopy GOT1. This suppression related domain found in other proteins and species (Mi et al., 2005). pattern suggested that Got1p physically interacts with components Based on these observations, we propose that Got1p influences of the COPII budding machinery; however, under the conditions membrane composition in a manner that can compensate for decreases of our co-immunoprecipitation experiments we failed to detect such in certain activities required for ER and/or Golgi transport. In the interactions, including any association with the Yip1 complex. We case of reduced Yip1 complex activity, elevated Got1p activity could 1548 Journal of Cell Science 122 (10)

change the ER lipid makeup in a way that facilitates COPII vesicle SDS-PAGE sample buffer and heated for 10 minutes at 37°C. Sucrose gradient budding. Our proposal predicts that the concentration of specific lipid fractionation of membrane organelles was performed as described (Powers and Barlowe, 1998). Cell extracts were loaded on top of 11-step 18-60% (wt/vol.) sucrose species in ER membranes will be altered depending on Got1p gradients and centrifuged at 164,000 ϫ g (SW40 rotor, Beckman Instruments, Palo expression level. A preliminary analysis of phospholipid species from Alto, CA) for 3 hours at 4°C. Eighteen 0.65 ml fractions were collected from the top whole cells indicated alterations in acyl chain length and degree of of the gradient and analyzed by immunoblot. Relative protein levels in each fraction Δ were quantified by densitometry using ImageJ (Rasband, 1997-2005). The plotted saturation when got1 cells were compared with wild-type cells optical density (OD) per fraction was calculated by dividing the OD of the fraction (unpublished). However, future experimentation will be necessary to over the total sum of ODs throughout the gradient for that particular protein. fully assess lipid composition in different subcellular compartments Velocity gradient sedimentation of detergent solubilized membrane proteins was when isolated from cells that have varying levels of Got1p. performed at 4°C (Price et al., 2000) with minor modification. Briefly, spheroplasts in lysis buffer (Baker et al., 1988) were disrupted in a Dounce homogenizer, cleared (1500 ϫ g, 5 minutes) and membranes sedimented (20,000 ϫ g, 5 minutes). Pellets Materials and Methods were resuspended with 1 ml of gradient buffer (GB: 25 mM HEPES pH 7.0, 100 Yeast strains and media mM KOAc) containing 1 mM DTT and membranes solubilized with addition of an Yeast strains used in this study are listed in supplementary material Table S1. Unless equal volume of 2% Triton X-100 prepared in GB. Solubilized extracts were cleared noted otherwise, cultures were grown at 25°C (for mutant stains) or 30°C (for wild- (100,000 ϫ g, 10 minutes) and 1.4 ml supernatant fluid was loaded on top of a 10.4 type strains) in rich medium (YPD: 1% Bacto yeast extract, 2% Bacto peptone, 2% ml 1-12% continuous sucrose gradient prepared in GB with 1% Triton X-100. dextrose), or in minimal medium (YMD: 0.7% USBiological yeast nitrogen base Molecular mass markers (beef pancreas ribonuclease A, rabbit muscle aldolase, and without amino acids, 2% dextrose and appropriate amino acid supplements). Standard beef liver catalase; Amersham Biosciences) were loaded on parallel gradients, and yeast (Sherman, 1991) and bacterial (Ausubel et al., 1987) molecular biology methods all were centrifuged at 175,000 ϫ g (SW41 rotor, Beckman Instruments) for 24 hours. were used. Yeast cells were transformed by the lithium acetate method (Elble, 1992). Twelve fractions were collected from the top of the gradient and the last fraction was Plasmids pRS426-FYV8, pRS426-TSC3, pRS425-GOT1, pRS426-GOT1, pRS426- used to resuspend the pellet. Relative levels of proteins and sucrose concentration of SFT2, pRS426-YIP1 and pRS426-YIF1 were generated by amplification of genomic individual fractions were determined as above. DNA obtained from FY834 (primers available upon request). The GAL1-3HA-GOT1 strain was constructed in CBY740 as described (Longtine et al., 1998). The Immunoprecipitations R R KAN MX6 marker of CBY1574 was exchanged to NAT MX4 (Tong et al., 2001) and For immunoprecipitation of Yif1p-3HA, 0.3 ml microsomes (~1 mg/ml membrane then mated with CBY2087 to generate CBY1769. Wild-type (CBY740) and GAL1- protein) were solubilized in an equal volume of 0.5% digitonin in buffer 88-8 (Kuehn 3HA-GOT1 (CBY2577) strains were transformed pJK59 [CEN SEC63-GFP URA3] et al., 1998) in the presence of 10 mM PMSF and 5 mM EDTA. After centrifugation (Prinz et al., 2000), or a chromosomal GFP tag was introduced into the SEC7 locus at 20,000 ϫ g for 4 minutes at room temperature, the supernatant fluid (~0.5 ml) as described (Rossanese et al., 2001) to generate CBY2537, CBY2542, CBY2538 was transferred to a fresh tube on ice. The solubilized material was diluted with 1.5 and CBY2543, respectively. volumes of 0.05% digitonin/buffer 88-8 and proteins immunoprecipitated for 2 hours at 4°C by addition of 2 μg anti-HA antibody and 25 μl of 20% protein-A-Sepharose Antibodies and immunoblotting beads (Amersham Biosciences). After washing beads, bound protein was released Polyclonal antibodies were raised against a GST-Got1p fusion protein with the by addition of 0.03 ml of SDS-PAGE sample buffer and heated at 75°C for 3 minutes. cytosolic C-terminus of Got1p (amino acid positions 109-138) expressed from plasmid For immunoprecipitation of Got1p-3MYC, microsomes as above were solubilized pGEX-2T. The fusion protein was purified according to the manufacturer’s on ice for 20 minutes with IP-buffer (150 mM NaCl, 15 mM Tris-HCl, pH 7.5, 1% specifications (Amersham Biosciences) and used to immunize rabbits by standard Triton X-100, 2 mM NaN3) in the presence of 2.5 mM EDTA, 5 mM PMSF, 0.5 μM procedures (Covance). For western blots, anti-Got1p serum was diluted 1:500. Leupeptin and 2.6 μM Pepstatin A. Samples were processed as above and supernatants Antibodies against CPY (Rothblatt et al., 1989), Sec61p (Stirling et al., 1992), Kar2p diluted 2.6-fold with IP buffer to a final volume of 1.2 ml. Anti-Myc antibody (4 μg) (Brodsky and Schekman, 1993), Sec12p (Powers and Barlowe, 1998), Sec23p (Hicke and protein-A-Sepharose beads (30 μl of 20% solution) were added and proteins and Schekman, 1989), Sec24p (Hicke et al., 1992), Sec13p (Salama et al., 1993), immunoprecipitated for 3 hours at 4°C. After washes, bound protein was released Och1p, Erv41p (Otte et al., 2001), Yip1p (Heidtman et al., 2003), Yif1p (Matern et from the beads by the addition of 25 μl of SDS-PAGE sample buffer and heated at al., 2000), Yos1p (Heidtman et al., 2005), Erv25p (Belden and Barlowe, 1996), Erv26p

Journal of Cell Science 37°C for 10 minutes. (Bue et al., 2006), Ypt1p (Rexach et al., 1994), Ypt7p (Haas et al., 1995), Gdi1p (Garrett et al., 1994), Uso1p (Ballew et al., 2005), Sec22p (Liu and Barlowe, 2002), Bet1p, Bos1p (Sogaard et al., 1994), Sed5p (Cao et al., 1998) and actin (Eitzen et Microscopy al., 2002) have been described. Monoclonal anti-HA (Sigma, clone HA-7) and anti- Fluorescent and bright-field images were acquired using a microscope (model BX51; ϫ cMyc (Covance, 9E10) were used. Western blot analysis was performed with Olympus) equipped with a 100 W mercury arc lamp, Plan Apochromat 60 objective nitrocellulose membranes or PVDF membranes (for detection of Yos1p and Got1p), (1.4 NA) and a Sensicam QE CCD camera (Cooke). The camera and microscope using the SuperSignal West Pico chemiluminiscent substrate (Pierce Chemical) and were controlled by the IP Lab system (Scanalytics). All images were processed in developed on film or with a UVP Bioimaging System. Openlab software (Improvision, Lexington, MA). To quantify ER morphologies, cells expressing Sec63-GFP were scored for the appearance of elaborated and discontinuous Multicopy suppressor screen or gapped ER structures within focal planes near the center of cells. To assess Golgi The multicopy suppression screen of yip1-2 (strain YXY12α) was performed as morphologies, cells expressing Sec7-GFP were monitored for the appearance of diffuse described (Heidtman et al., 2005). Briefly, ~10,000 transformants from a yeast genomic fluorescence distributions and loss of punctate structures. YEp24 library (Carlson and Botstein, 1982) were grown on YMD-Ura plates at 25°C. Colonies were then replica plated to YPD plates and incubated for 48 hours at 36°C. We thank John Flanagan, Joshua Wilson, Polina Shindiapina, Eighteen temperature resistant transformants were transferred to YMD-Ura plates, Christopher Hickey and Russell Monds for scientific discussions, Duane plasmid DNA isolated and retransformed into yip1-2 to test for plasmid-linked Compton and Bill Wickner for making their microscopes available and suppression. Of the 18 isolated plasmid preparations, 13 conferred varying levels of suppression upon retransformation. Plasmids inserts were sequenced with the primers Amity Manning and Polina Shindiapina for help with microscopy. All YEpF1 and YEpR3 (Heidtman et al., 2005). those mentioned are from Dartmouth Medical School. This work was supported by the National Institutes of Health. Deposited in PMC for In vitro vesicle budding, tethering, and transport assays release after 12 months. Yeast semi-intact cell membranes from wild-type and mutant strains were prepared as described (Baker et al., 1988). Vesicle budding, tethering and fusion assays using [35S]gpαf were performed as described (Barlowe, 1997; Cao et al., 1998). Data points References are the average of duplicate determinations and the error bars represent the range. Ausubel, R. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. To measure protein packaging into COPII vesicles, microsomes were prepared A. and Struhl, K. (1987). Current Protocols in Molecular Biology. New York: Greene (Wuestehube and Schekman, 1992), and preparative scale budding reactions performed Publishing and Wiley-Interscience. as described (Otte et al., 2001). Baetz, K. K., Krogan, N. J., Emili, A., Greenblatt, J. and Hieter, P. (2004). The ctf13- 30/CTF13 genomic haploinsufficiency modifier screen identifies the yeast chromatin remodeling complex RSC, which is required for the establishment of sister chromatid Subcellular fractionation and sucrose velocity gradient cohesion. Mol. Cell. Biol. 24, 1232-1244. sedimentation Baker, D., Hicke, L., Rexach, M., Schleyer, M. and Schekman, R. (1988). Reconstitution ER (P13) and Golgi (P100) membrane fractions were collected by differential of SEC gene product-dependent intercompartmental protein transport. Cell 54, 335-344. centrifugation of cell lysates prepared from wild-type and sec12-4 strains as described Ballew, N., Liu, Y. and Barlowe, C. (2005). A Rab requirement is not bypassed in SLY1- (Belden and Barlowe, 2001). Membrane pellets were resuspended with 0.05 ml of 20 suppression. Mol. Biol. Cell 16, 1839-1849. GOT1 in ER-Golgi transport 1549

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