PMC20140719ascii.key2133178The Journal of Cell Biologysurname:Gimeno;given-names:Ruthendoplasmic reticulum transport vesicle COPII proton-ATPase Saccharomyces cerevisiaesurname:Kaiser;given-names:Chris A.surname:Espenshade;given-names:Peter10330397surname:Crotwell;given-names:Michellesurname:Roberg;given-names:Kevin J.414521331781999front65967200LST1 Is a SEC24 Homologue Used for Selective Export of the Plasma Membrane ATPase from the Endoplasmic ReticulumGeneLST1GeneSEC24GenePlasma Membrane ATPaseabstract114114In Saccharomyces cerevisiae, vesicles that carry proteins from the ER to the Golgi compartment are encapsulated by COPII coat proteins.GeneCOPII coat proteins252We identified mutations in ten genes, designated LST (lethal with sec-thirteen), that were lethal in combination with the COPII mutation sec13-1.GeneLSTGenelethal withGeneCOPII mutation sec13-1400LST1 showed synthetic-lethal interactions with the complete set of COPII genes, indicating that LST1 encodes a new COPII function.GeneLST1GeneCOPII genesGeneLST1532LST1 codes for a protein similar in sequence to the COPII subunit Sec24p.GeneLST1608Like Sec24p, Lst1p is a peripheral ER membrane protein that binds to the COPII subunit Sec23p.GeneSec24pGeneLst1p704Chromosomal deletion of LST1 is not lethal, but inhibits transport of the plasma membrane proton-ATPase (Pma1p) to the cell surface, causing poor growth on media of low pH.GeneLST1Geneplasma membrane proton-ATPaseGenePma1p879Localization by both immunofluorescence microscopy and cell fractionation shows that the export of Pma1p from the ER is impaired in lst1Delta mutants.GenePma1pGenelst1Delta mutants1033Transport of other proteins from the ER was not affected by lst1Delta, nor was Pma1p transport found to be particularly sensitive to other COPII defects.GenePma1p transport1188Together, these findings suggest that a specialized form of the COPII coat subunit, with Lst1p in place of Sec24p, is used for the efficient packaging of Pma1p into vesicles derived from the ER.GeneCOPII coat subunitGeneLst1pGeneSec24pGenePma1pparagraph13811381The plasma membrane proton-ATPase (Pma1p)1 is an essential integral membrane protein that couples ATP hydrolysis to the translocation of protons across the plasma membrane (Serrano et al., 1986).Geneplasma membrane proton-ATPaseGenePma1p1580The proton gradient generated by Pma1p then drives the uptake of nutrients, such as amino acids, from the extracellular medium (Vallejo and Serrano, 1989).GenePma1p1737A second physiological function of Pma1p is to maintain the cytosol at a neutral pH.GenePma1p1823In medium of low pH, the growth rate is limited by the amount of cellular Pma1p (McCusker et al., 1987; Portillo and Serrano, 1989).1957Pma1p transport to the cell surface depends upon the secretory pathway defined by the sec genes (Brada and Schekman, 1988; Chang and Slayman, 1991).GenePma1p2109Pma1p is one of the most abundant cargo molecules of the secretory pathway, constituting 25-50% of the total plasma membrane protein (Serrano, 1991).GenePma1p2261Because of its abundance and physiological importance, one might expect that yeast cells would have specialized mechanisms to ensure efficient transport of Pma1p through the secretory pathway.GenePma1p2456Such a function has been suggested for two proteins, Ast1p and Ast2p, in the transport of Pma1p from the Golgi compartment to the plasma membrane (Chang and Fink, 1995).GeneAst1pGeneAst2pGenePma1p2629For early steps in the secretory pathway, proteins that are specifically required for the transport of Pma1p have not yet been identified.GenePma1pparagraph27722772Proteins destined for the plasma membrane are transported from the ER to the Golgi compartment by vesicles coated with a set of proteins known as COPII (Barlowe et al., 1994).2950These COPII coats are thought to cause the deformation of the membrane into a vesicle and to recruit cargo molecules into vesicle buds (reviewed by Schekman and Orci, 1996).GeneCOPII coats3126The stepwise recruitment and assembly of the COPII coat onto the membrane is thought to occur as follows.3234Action of the ER resident membrane protein Sec12p, a guanine nucleotide exchange factor for Sar1p, causes Sar1p to bind to the ER membrane (Barlowe and Schekman, 1993).GeneER resident membrane protein Sec12pGeneguanine nucleotide exchange factorGeneSar1pGeneSar1p3406Membrane-associated Sar1p, in turn, recruits the soluble Sec23p/Sec24p and Sec13p/Sec31p complexes (Matsuoka et al., 1998).GeneSar1pGeneSec23pGeneSec24pGeneSec13pGeneSec31p3530Sec16p resides on the ER membrane and binds to both the Sec23p/Sec24p and Sec13p/Sec31p complexes, likely organizing their assembly onto the membrane (Espenshade et al., 1995; Gimeno et al., 1996; Shaywitz et al., 1997).GeneSec16pGeneSec23pGeneSec24pGeneSec13pGeneSec31p3755To examine the role of different COPII coat subunits in recruitment of cargo molecules to vesicles, partially assembled COPII complexes have been tested for their ability to associate with cargo proteins.GeneCOPII coat subunits3963Association of a membrane-bound complex of Sar1p and Sec23p/Sec24p with integral membrane proteins indicates that cargo proteins may laterally partition into the vesicle membrane by virtue of their affinity for the Sec23p/Sec24p protein complex (Aridor et al., 1998; Kuehn et al., 1998).GeneSar1pGeneSec23pGeneSec24pGeneintegral membrane proteinsGeneSec23pGeneSec24pparagraph42554255An early indication that the COPII coat subunits would physically interact came from specific genetic interactions between mutations in COPII genes.GeneCOPII coat subunitsGeneCOPII genes4406When temperature-sensitive mutations in COPII genes are combined, the resulting double mutants are almost always more growth- restrictive than the component single mutations, and are usually inviable at 24 C.GeneCOPII genes4616These synthetic-lethal interactions are restricted to genes involved in COPII vesicle formation and do not occur when mutations in genes required for vesicle formation are combined with genes required for vesicle fusion (Kaiser and Schekman, 1990).4867The specificity of this type of genetic interaction suggested that synthetic lethality with known COPII mutations would be a useful criterion to identify new mutations involved in the assembly of the COPII coat.GeneCOPII coatparagraph50825082We screened for mutations that were lethal with the COPII mutation sec13-1 and identified ten LST genes (lethal with sec-thirteen).GeneCOPII mutation sec13-1Geneten LST genes5215As we describe elsewhere, most of the LST genes are related to an unanticipated role for SEC13 in the regulated delivery of specific amino acid permeases to the cell surface (Roberg et al., 1997a,b).GeneLST genesGeneSEC135417Accordingly, these LST genes display synthetic-lethal interactions with SEC13, but not with the other COPII genes.GeneLST genesGeneSEC13GeneCOPII genes5533On the other hand, mutations in LST1 were lethal with the full set of mutations defective in COPII vesicle budding, but not with mutations defective in vesicle fusion, indicating that LST1 does participate in vesicle budding at the ER.GeneLST1GeneLST15772Here we show that LST1 encodes a homologue of the COPII subunit, Sec24p, and that Lst1p is a peripheral membrane protein localized to the ER that can form complexes with Sec23p.GeneLST1GeneCOPII subunitGeneSec24pGeneLst1pGeneSec23p5952The LST1 gene is not essential, but by examination of the phenotypes of lst1Delta mutants we show that LST1 is required for the efficient export of Pma1p from the ER to the Golgi compartment.GeneLST1 geneGenelst1Delta mutantsGeneLST1GenePma1p6147These results suggest a specialized form of the vesicle coat that is responsible for recruitment of Pma1p into COPII-coated vesicles.GenePma1ptitle_162786278Materials and Methodstitle_263006300Media, Strains, and Plasmidsparagraph63296329The Saccharomyces cerevisiae strains used in this study are listed in Table I.6409Rich medium (YPD) and supplemented minimal medium (SMM) were prepared according to Kaiser et al. (1994).6515To evaluate growth at low pH, YPD was adjusted to pH 3.8 with HCl (this medium remained at pH 3.8 throughout the growth of a yeast culture).6658For some experiments, SMM was buffered to pH 6.5 using 50 mM MOPS and 50 mM MES.6740Genetic manipulations were performed according to standard protocols (Kaiser et al., 1994).6833DNA manipulations were carried out as described in Sambrook et al. (1989).6909pAF70 carries the SEC24 gene in the centromere vector pCT3 (URA3; Gimeno et al., 1996).GenepAF70GeneSEC24 gene6998pKR34 and pKR41 carry the 3.8-kb KpnI/SalI fragment containing the SEC24 gene from pAF70 in the 2mu vectors pRS426 (URA3) and pRS425 (LEU2), respectively.GenepKR34GenepKR41GeneKpnIGeneSalIGeneSEC24 geneGenepAF70GeneURA3GenepRS425GeneLEU27154pKR17 carries the LST1 gene on a 3.5-kb fragment in the centromere vector pRS316 (URA3).GenepKR17GeneLST1 gene7245A subclone of the LST1 gene from pKR17 into the 2mu vector pRS426 gave yeast transformants at a very low efficiency because of the toxicity of LST1 sequences when present at high copy.GeneLST1 geneGenepKR17GeneLST1 sequences7432To study the toxic effects of LST1, pKR35 was constructed which contains the entire LST1 coding sequence expressed from pGAL1 on pCD43 (URA3).GeneLST1GenepKR35GeneLST1 coding sequenceGenepGAL1GenepCD43GeneURA37577pKR35 will prevent growth under conditions of full induction on galactose medium, establishing that overexpression of Lst1p is toxic to yeast cells.GenepKR35GeneLst1p7728Under conditions of partial induction of pGAL1-LST1 in cells grown on raffinose, pKR35 will complement lst1Delta::LEU2 for growth on acidic medium.GenepGAL1-LST1GenepKR35 will complement lst1DeltaGeneLEU27877This shows that the LST1 open reading frame carried on pKR35 still posses LST1 function.GeneLST1GenepKR35GeneLST1paragraph79687968Epitope-tagged LST1 was constructed as follows.GeneLST18016First, the NotI site in the polylinker of pKR17 was deleted with a 350-bp SmaI/NaeI fragment (pKR17Delta), and then a 12-bp linker carrying a NotI site (1127; New England BioLabs) was inserted at the Eco47III site (at codon 13 of LST1) of pKR17Delta to make pKR17N.GeneNotI siteGenepKR17 was deleted with aGeneSmaIGeneNaeIGenepKR17DeltaGeneNotI siteGeneEco47III siteGenepKR17DeltaGenepKR17N8285pKR17HA carries the 100-bp NotI fragment from pGTEPI (Tyers et al., 1993), which encodes three tandem copies of the hemagglutinin (HA1) epitope, inserted into the NotI site of pKR17N.GenepKR17HAGeneNotIGenehemagglutininGeneHA1GeneNotI siteGenepKR17N8472Restriction analysis using sites flanking the point of insertion revealed that two 100-bp inserts (six HA epitopes) were present in pKR17HA.GeneHA epitopesGenepKR17HA8615pKR17HA was transformed into CKY536 to make CKY535 (MATa lst1Delta:: LEU2 leu2-3, 112 ura3-52 [pKR17HA]).GenepKR17HAGeneCKY536GeneCKY535GeneMATa lst1DeltaGeneLEU2 leu2-3, 112 ura3-52GenepKR17HAtitle_287138713Synthetic-lethal Screenparagraph87378737The following plasmids and strains were constructed for use in the sec13-1 synthetic-lethal screen.Genesec13-18838The plasmid pKR1 carries SEC13 on a 1.8-kb SalI/ BamHI fragment excised from pCK1313 (Pryer et al., 1993), inserted into pRS316 (Sikorski and Heiter, 1989).GeneSEC13GeneSalIGeneBamHI8996pKR4 carries the same 1.8-kb SalI/ BamHI fragment and a 3.8-kb NheI/BamHI fragment containing ADE3 from pDK255, both inserted into the vector pRS315 (Sikorski and Heiter, 1989).GenepKR4GeneSalIGeneBamHIGeneNheIGeneBamHIGeneADE39176CUY563 and CKY45 were crossed to produce a MATa ade2 ade3 leu2 ura3 sec13-1 segregant, which was transformed with pKR4 to give CKY423.GeneCUY563GeneCKY45GeneMATaGeneade2Geneade3Geneleu2Geneura3 sec13-1 segregantGenepKR4GeneCKY4239313The mating type of CKY423 was switched by ectopic expression of the HO gene (Herskowitz and Jensen, 1991) to give CKY424.GeneCKY423GeneHO geneGeneCKY424paragraph94369436Cultures of CKY423 and CKY424 were mutagenized by irradiation with a germicidal UV lamp at a dose resulting in 10% cell survival.GeneCKY423GeneCKY4249567Mutagenized cells were plated on YPD at a density of 150 colonies per plate.9645After 5 d of growth at 24 C, colonies with a solid red color and no white sectors were selected for further analysis.9764The dependence of the nonsectoring phenotype on the sec13-1 mutation was tested by transforming candidate mutants with either pKR1 or pRS316.Genesec13-19906Strains that sectored after transformation with pKR1, but not after transformation with pRS316, were scored as sec13-1-dependent.GenepKR1GenepRS316Genesec13-1paragraph1004110041Complementation tests were performed by mating mutants isolated from CKY423 with those isolated from CKY424.GeneCKY423GeneCKY42410151Zygotes isolated by micromanipulation were scored for their ability to form sectored colonies on YPD plates.10261The genes defined by these complementation groups were designated LST.GeneLST10333All lst mutant strains were backcrossed to a parental strain twice.paragraph1040210402The lst sec13-1 double mutants were converted to lst single mutants by integration of a wild-type copy of SEC13 at the sec13-1 locus, using the integrating plasmid p1312 (SEC13 URA3; Pryer et al., 1993).Genesec13-1 double mutantsGeneSEC13Genesec13-1 locusGenep1312GeneSEC13 URA310607The integrants were grown on YPD and cells from white sectors (indicating loss of pKR4) were isolated.GenepKR410712The integration of a wild-type copy of SEC13 was confirmed by the ability of the cells from white sectors to grow at 36 C, a temperature that is restrictive for the sec13-1 mutation.GeneSEC13Genesec13-110897Owing to the poor growth of lst9 strains, we were not able to construct a lst9 single mutant by this method.Genelst9Genelst9 single mutantparagraph1100911009To test for synthetic-lethal interactions between lst mutations and mutations in sec genes, lst mutants CKY435 (lst1-1), CKY436 (lst2-1), CKY437 (lst3-1), CKY438 (lst4-1), CKY439 (lst5-1), CKY440 (lst6-1), CKY441 (lst7-1), and CKY442 (lst8-1) were crossed to the sec mutants CKY45 (sec13-1), CKY50 (sec16-2), CKY78 (sec23-1), and CKY450 (sec31-2).Genelst mutants CKY435Genelst1-1GeneCKY436Genelst2-1GeneCKY437Genelst3-1GeneCKY438Genelst4-1GeneCKY439Genelst5-1GeneCKY440Genelst6-1GeneCKY441Genelst7-1GeneCKY442Genelst8-1Genesec mutants CKY45Genesec13-1GeneCKY50Genesec16-2GeneCKY78Genesec23-1GeneCKY450Genesec31-211360Inviability of a given lst sec double mutant was inferred from crosses where lethality segregated as a two-gene trait (most tetrads giving a segregation pattern of 1:3 for lethality), an outcome that was easily detectable since crosses to wild-type typically gave >95% spore viability.Genelst sec double mutant11649The segregation pattern of the sec mutation in the surviving sister spores was used as an additional test to establish that the inviable spores always carried the sec mutation, and therefore were not the result of random spore death.title_21188411884Construction of lst1Delta Mutantsparagraph1191511915Replacement of the chromosomal LST1 gene with an allele disrupted with the LEU2 gene was constructed as follows.Genechromosomal LST1 geneGeneLEU2 gene12029pKR18 carries the 5' half of LST1 on a 2.0-kb Xho1/HindIII fragment inserted into pRS316.GenepKR18GeneLST1GeneXho1GeneHindIII12120A 2.0-kb HindIII/BamHI fragment containing the LEU2 gene from plasmid pJJ252 (Jones and Prakash, 1990) and a 250-bp BclI/SacI fragment from the 3' noncoding region of LST1 were inserted into pKR18 to construct pKR28.GeneHindIIIGeneBamHIGeneLEU2 geneGeneBclIGeneSacIGeneLST1GenepKR1812341The NH2-terminal coding region of LST1 (except for codons 1-13) was removed by deleting a 1.7-kb Eco47III/MscI fragment from pKR28 to generate pKR28Delta.GeneLST1GeneEco47IIIGeneMscIGenepKR28GenepKR28Delta12496The lst1Delta::LEU2 construct, liberated from pKR28Delta by digestion with XhoI, was transformed into the wild-type diploid strain CKY348 (MATa/alpha leu2-3,112/leu2-3,112 ura3-52/ura3-52).GeneLEU2 constructGenepKR28DeltaGeneXhoIGeneCKY348GeneMATa/alpha leu2-3,112/leu2-3Gene112Geneura3-52Geneura3-5212687On sporulation and dissection, this diploid gave four viable spore clones, and haploid segregants carrying lst1Delta::LEU2 were confirmed by Southern blotting.Genelst1DeltaGeneLEU212848One such segregant was further backcrossed to our S288C genetic background to give strains CKY536 and CKY542.GeneCKY542title_21295112951Proton Efflux from Intact Yeast Cellsparagraph1298912989Pma1p activity was assayed by proton efflux from intact cells into the external medium.GenePma1p13077Cells were grown to exponential phase in YPD at 37 C, washed, and then stored in deionized water at 4 C overnight.13193Cell number was measured by light scattering, and a total of 25 A600 units (~5 x 108 cells) was suspended in 5 ml of 100 mM KCl, 10 mM glycine, pH 4.0.13347The pH of the cell suspension was measured using a combination electrode at 25 C with constant stirring.13454Once the pH had stabilized (~10 min), glucose was added to a final concentration of 40 mM and the ensuing drop in pH was recorded at 30-s intervals over 15 min.13616In comparison of wild-type (CKY443) and lst1Delta (CKY536) strains, both suspensions had identical cell concentration as measured by light scattering, and showed the same response to calibration pulses with HCl.Genewild-type (CKY443)Genelst1DeltaGeneCKY536title_21383513835Immunofluorescence Microscopyparagraph1386513865The intracellular location of Pma1p in wild-type (CKY443) and lst1Delta (CKY536) cells was examined by indirect immunofluorescence microscopy using techniques described previously (Pringle et al., 1991; Espenshade et al., 1995).GenePma1pGenelst1DeltaGeneCKY53614095Strains were grown exponentially in SMM medium, pH 7.2, at 30 C.14161Cells were fixed in 3.7% formaldehyde and then converted to spheroplasts by digestion with lyticase.Genelyticase14263Both primary and secondary antibody incubations were for 1 h at 25 C.14333Affinity-purified anti-Pma1p antibody was prepared as follows.Geneanti-Pma1p antibody14396A crude preparation of yeast membranes was resolved by preparative SDS-PAGE, and after transfer of proteins to a nitrocellulose membrane by electrophoresis, the strip of membrane that contained Pma1p was excised.GenePma1p14612Rabbit antiserum to Pma1p was applied to the nitrocellulose strip, and after the strip was washed with 20 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Tween 20, the bound antibody was eluted with 100 mM glycine, pH 2.8, 500 mM NaCl, 0.5% Tween 20.GenePma1p14853Affinity-purified Pma1p was used at a 1:100 dilution and FITC-conjugated anti-rabbit IgG was used at 1:200 dilution.GenePma1pGeneanti-rabbit IgG14971Mounting medium was supplemented with 4',6-diamidino-2-phenylindole (DAPI).15048Micrographs were taken with a Nikon Eclipse TE300 microscope with a Hamamatsu Orca C4742-95 CCD camera.paragraph1515515155For the localization of Lst1p-HA, CKY535 was grown on SMM to exponential phase and prepared as described above.GeneLst1p-HAGeneCKY53515267For visualization of Lst1p-HA, the 12CA5 antibody (Berkeley Antibody Co., Inc.) was used at a 1:5,000 dilution and FITC-conjugated goat anti-mouse IgG was used at a 1:50 dilution.GeneLst1p-HAGeneanti-mouse IgG15450Rabbit anti-Kar2p polyclonal serum (a gift of M. Rose, Princeton University, Princeton, NJ) was used at a 1:1,000 dilution and rhodamine-conjugated goat anti-rabbit IgG was used at a 1:200 dilution.GeneRabbit anti-Kar2pGeneanti-rabbit IgG15652Samples were viewed and imaged using a Nikon Optiphot 2 microscope and a Photometric ImagePoint CCD camera.15761Images were recorded using IP-Lab software (Molecular Dynamics, Inc.).title_21583915839Cell Fractionationparagraph1585815858Cell organelles were fractionated on equilibrium density gradients as previously described (Roberg et al., 1997a).15973Cultures were grown exponentially at 24 C and then shifted to 37 C for 3 h.160491.6 x 109 cells were collected by centrifugation and suspended in 0.5 ml STE10 (10% [wt/wt] sucrose, 10 mM Tris-HCl, pH 7.6, 10 mM EDTA) with a protease inhibitor cocktail (1 mM PMSF, 0.5 mug/ml leupeptin, 0.7 mug/ml pepstatin, 2 mug/ml aprotinin) and lysed by vortexing with glass beads.Genepepstatin, 2 mug/ml aprotinin16340An additional 1 ml of STE10 was added, and the lysate was cleared of unbroken cells and large cell debris by centrifugation at 300 g for 2 min.GeneSTE1016485The cleared extract (300 mul) was layered on top of a 5-ml, 20-60% linear sucrose gradient in TE (10 mM Tris-HCl, pH 7.6, 10 mM EDTA) prepared for an SW50.1 rotor (Beckman Instruments, Inc.).16679Samples were centrifuged 100,000 g for 18 h at 4 C and fractions of 300 mul were collected from the top of the gradient.16801Protein was precipitated from each fraction by the addition of 100 mul of 0.15% deoxycholate and 100 mul of 72% trichloroacetic acid.16937Protein pellets were collected by centrifugation at 13,000 g, washed with cold acetone, and then solubilized in ESB (60 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 10% glycerol, 0.02% bromophenol blue).17140Pma1p, Gas1p, and Sec61p were resolved by SDS-PAGE and were detected by immunoblotting.GenePma1pGeneGas1pGeneSec61p17229The relative amount of each protein in cell fractions was determined by densitometry using an Ultroscan 2202 (LKB Instruments, Inc.).17363The Golgi GDPase activity was assayed in gradient fractions before protein precipitation using standard methods (Abeijon et al., 1989).GeneGolgi GDPaseparagraph1750617506The subcellular distribution of Lst1p-HA was examined using techniques described previously (Espenshade et al., 1995).GeneLst1p-HA17625CKY535 carrying pKR17HA, which expresses Lst1p-HA, was grown to exponential phase in SMM without uracil.GeneCKY535GenepKR17HAGeneLst1p-HA177322 x 109 cells were harvested, converted to spheroplasts, and then gently lysed by glass beads in 500 mul of cell lysis buffer (20 mM MES, pH 6.5, 100 mM NaCl, 5 mM MgCl2) including the protease inhibitor cocktail.17948The cell extract was sequentially centrifuged at 500 g for 20 min, 10,000 g for 20 min, and 150,000 g for 60 min, to give one soluble and three particulate fractions.paragraph1811718117Release of Lst1p-HA from the particulate fraction was examined by treating cell extracts with 500 mM NaCl, 100 mM sodium carbonate, pH 11.5, 2.5 M urea, or 1% Triton X-100.GeneReleaseGeneLst1p-HA18292After 1 h of incubation at 4 C, samples were centrifuged at 50,000 g for 30 min to separate soluble and particulate fractions.18419Fractions from both experiments were solubilized in sample buffer and analyzed by immunoblotting.title_21851818518Immunoblottingparagraph1853318533Samples of 10-30 mul in ESB were resolved by SDS-PAGE and immunoblotting was conducted according to standard protocols (Harlow and Lane, 1988).18678For transfer of Lst1p to nitrocellulose membranes, 0.1% SDS was included in the transfer buffer.GeneLst1p18776The following antibodies were used: mouse monoclonal 12CA5 anti-HA at 1:1,000 dilution; rabbit anti-Pma1p (a gift of A. Chang, Albert Einstein College of Medicine, Bronx, NY) at 1:500 dilution; rabbit anti-Gas1p (a gift of H. Riezman, University of Basel, Basel, Switzerland) at 1:10,000 dilution; rabbit anti-Sec61p (a gift of R. Schekman, University of California, Berkeley, CA) at 1:3,000 dilution; rabbit anti-Gdh2p (a gift of B. Magasanik, Massachusetts Institute of Technology, Cambridge, MA) at 1:1,000 dilution; HRP-coupled sheep anti-mouse Ig and HRP-coupled sheep anti-rabbit Ig (Nycomed Amersham Corp.) at 1:10,000 dilution.Genemouse monoclonal 12CA5 anti-HAGenerabbit anti-Pma1pGenerabbit anti-Gas1pGenerabbit anti-Sec61pGenerabbit anti-Gdh2pGeneHRP-coupled sheep anti-mouse Ig and HRP-coupled sheep anti-rabbit Ig19420Blots were developed using chemiluminescence detection system (Nycomed Amersham Corp.).title_21951419514Pulse-Chase Kinetics of Invertase MaturationGeneInvertaseparagraph1956119561The strains used for radiolabeling all carried the plasmid pNV31, which carries the SUC2 gene under the constitutive TPI1 promoter (a gift of M. Lewis, Medical Research Council Laboratories of Molecular Biology, Cambridge, UK).GeneSUC2 geneGeneTPI1 promoter19792Wild-type (CKY540) and lst1Delta (CKY542) strains were grown in SMM without methionine (buffered with 50 mM MES and 50 mM MOPS to pH 6.5) at 24 C to exponential phase, and then shifted to 37 C for 3 h before labeling.GeneWild-type (CKY540)Genelst1DeltaGeneCKY54220013A sec12-4 strain (CKY541) was similarly grown to exponential phase at 24 C, but was shifted to 37 C 5 min before the addition of label.Genesec12-4 strainGeneCKY54120150Radiolabeling and immunoprecipitation of invertase was performed as previously described (Gimeno et al., 1995; Elrod-Erickson and Kaiser, 1996).Geneinvertasetitle_22029820298Two-Hybrid Interactionsparagraph2032220322The yeast two-hybrid assay was used to test potential protein-protein interactions as previously described (Gyuris et al., 1993; Bartel and Fields, 1995).20478Interactions were tested between either Lst1p or Sec24p fused to the LexA DNA-binding domain and Sec23p fused to an acidic transcriptional activation domain.GeneLst1pGeneSec24pGeneLexA DNA-binding domainGeneSec23p20637The following plasmids were used: pPE81 carries SEC23 fused to the acidic activation domain of pJG4-5 (Espenshade et al., 1995); pRH286 carries SEC24 (codons 34-926) fused to the lexA DNA-binding domain in pEG202 (Gimeno et al., 1996); pKR37 carries LST1 fused to the lexA DNA-binding domain in pGilda (a derivative of pEG202 with pGAL1; provided by D. Shaywitz).GenepPE81GeneSEC23GenepJG4-5GeneSEC24GenelexA DNA-binding domainGenepKR37GeneLST1GenelexA DNA-binding domainparagraph2100921009Combinations of control and fusion protein plasmids, along with the reporter plasmid pSH18-34, were transformed into the strain EGY40 (Golemis and Brent, 1992).21170Strains were grown exponentially in SMM with 2% raffinose as the carbon source.21251Galactose was added to a concentration of 2%, and incubation was continued for 10 h to induce fusion proteins expressed from pGAL1.GenepGAL121384Assays for beta-galactosidase activity were performed on cells lysed by disruption with glass beads (Rose and Botstein, 1983).Genebeta-galactosidase21513Activity was normalized to total protein determined by the Bradford assay (Bio-Rad Laboratories).title_22160921609Binding of Lst1p to Sec23pGeneLst1pGeneSec23pparagraph2163621636A gene fusion expressing Lst1p fused to glutathione S-transferase (GST) was constructed by inserting the 3.0-kb BamHI/XhoI fragment of pKR17HA into pRD56 (a gift of R. Deshaies, California Institute of Technology, Pasadena, CA) to construct pRH254, which gives GST-Lst1p-HA (amino acids 14-927 of Lst1p) fusion expressed from pGAL1.Genegene fusionGeneLst1pGeneglutathioneGeneGSTGeneBamHIGeneXhoIGenepKR17HAGenepRD56GenepRH254GeneGSTGeneLst1pGeneHAGeneLst1pGenepGAL121972pPE123 is the SEC23 gene expressed from pGAL1 in pRS315 (Gimeno et al., 1996).GenepPE123GeneSEC23 geneGenepGAL1GenepRS31522053Binding interactions were tested from extracts of CKY473 transformed with pRH254 (GST-Lst1p-HA) and either pCD43 (vector) or pPE123 (Sec23p).GeneCKY473GenepRH254GeneGSTGeneLst1pGenepPE123GeneSec23pparagraph2220222202Cells were grown to exponential phase in SMM with 2% raffinose, galactose was added to 2%, and incubation was continued for 2 h at 30 C to induce pGAL1 expression.GenepGAL1223675 x 108 cells were converted to spheroplasts as previously described (Espenshade et al., 1995) and then gently lysed using glass beads in IP buffer (20 mM Hepes-KOH, pH 6.8, 80 mM KOAc, 5 mM magnesium acetate, 0.02% Triton X-100) containing the protease inhibitor cocktail.22644The extract was diluted to 1 ml with IP buffer, and membranes were collected by centrifugation at 500 g for 20 min.22760This pellet was extracted with 1 ml of IP buffer and 600 mM NaCl for 10 min at 0 C to release membrane-bound protein complexes.22889After clarification by centrifugation at 90,000 g for 10 min, the extract was diluted threefold with IP buffer, and a 1-ml aliquot was removed and incubated at room temperature for 1 h with glutathione Sepharose 4B beads (Pharmacia Biotech, Inc.).23139The beads were washed twice with 200 mM NaCl, 20 mM Hepes-KOH, pH 6.8, 80 mM KOAc, 5 mM magnesium acetate, 0.02% Triton X-100, and once in IP buffer without Triton X-100.23311Proteins were released from glutathione Sepharose 4B beads by solubilization in ESB.Geneglutathione Sepharose 4B23397Samples of total lysate were prepared by adding 2x ESB to an equal amount of the diluted extract from the salt washed membranes.23528Samples were analyzed by immunoblots probed with anti-Sec23p antibody.Geneanti-Sec23p antibodyparagraph2360423604For analysis of the membrane association of GST-Lst1p-HA and Sec23p, cells expressing GST-Lst1p-HA, Sec23p, or both GST-Lst1p-HA and Sec23p from pGAL1, were grown in 2% raffinose and then induced by the addition of 2% galactose as described above.GeneGSTGeneLst1pGeneHAGeneSec23pGeneGSTGeneLst1pGeneHAGeneSec23pGeneGSTGeneLst1pGeneHAGeneSec23p from pGAL1238552 h after induction, 2 x 107 cells were collected by centrifugation and resuspended in 20 mul of cell lysis buffer (20 mM MES, pH 6.5, 100 mM NaCl, 5 mM MgCl2) with protease inhibitor cocktail.24051Cells were lysed by vigorous agitation with glass beads and an additional 500 mul of lysis buffer was added.24161The lysate was cleared of unlysed cells and large cell debris by centrifugation at 300 g for 3 min.2426250 mul of the supernatant was reserved for a total extract sample and the remainder was centrifuged to pellet ER membranes at 10,000 g for 30 min at 4 C in a microcentrifuge.24440An equal number of cell equivalents of total extract, membrane-pellet, and supernatant fractions was solubilized in ESB and analyzed by immunoblotting.24593The cytosolic protein Gdh2p was found only in the soluble fractions, demonstrating cell lysis was complete (data not shown).Genecytosolic protein Gdh2ptitle_12472624726Resultstitle_22473424734Mutations Synthetically Lethal with sec13-1Genesec13-1paragraph2477824778To find new genes required for the budding of COPII vesicles, we screened for mutations that displayed synthetic lethality with the COPII mutation sec13-1 using a plasmid sectoring assay (Roberg et al., 1997b).GeneCOPII mutation sec13-124991Strain CKY423 has the chromosomal mutations ade2 ade3 sec13-1 and harbors the plasmid pKR4, which carries wild-type copies of SEC13 and ADE3.Geneade2 ade3 sec13-1GeneSEC13GeneADE325136This strain accumulates a red pigment because of the ade2 mutation, but the spontaneous loss of pKR4 during the growth of a colony gives white sectors of ade2 ade3 segregants.Geneade2 mutationGenepKR4Geneade2 ade3 segregants25315In this strain, a mutation that is lethal with sec13-1 will produce a nonsectoring colony.Genesec13-125406Mutagenesis of CKY423 and the isogenic strain of opposite mating type, CKY424, yielded 139 nonsectoring mutants (Fig. 1).GeneCKY423GeneCKY42425530These strains were then tested for restored ability to sector after transformation with pKR1, which carries wild-type SEC13, but lacks the ADE3 gene.GenepKR1GeneADE3 gene25682By this test, 57 of the mutants had synthetic-lethal mutations that could be rescued by wild-type SEC13.25789In backcrosses, 52 mutants gave a segregation pattern indicating that the trait was due to a single nuclear mutation (Fig. 1).GeneFig. 1paragraph2591825918Matings between mutants identified 11 complementation groups using colony sectoring of the diploid as the criterion for allelic complementation.26063These complementation groups were designated LST (Table II).26124One of the complementation groups was shown to comprise recessive lethal mutations in the SEC13 gene itself (Roberg et al., 1997b).GeneSEC13 gene26259Tests for rescue of the nonsectoring phenotype by plasmids carrying known sec genes showed that LST10 was SEC16 (Roberg et al., 1997b).GeneLST10GeneSEC16title_22639826398Synthetic Interactions of lst Mutationsparagraph2643826438To perform further genetic tests on the lst mutations, the lst sec13-1 double mutants were converted to lst single mutants by integration of a wild-type copy of SEC13 at the sec13-1 locus (Materials and Methods).Genesec13-1 double mutantsGeneSEC13Genesec13-1 locus26653Representative lst single mutants were then crossed to sec16, sec23, and sec31 mutants.Genesec16Genesec23Genesec31 mutants26743For mutations in LST2, LST3, LST4, LST5, LST7, and LST8, only crosses to sec13-1 gave a segregation pattern indicative of a synthetic-lethal interaction (Table III).GeneLST2GeneLST3GeneLST4GeneLST5GeneLST7GeneLST8Genesec13-126911We have subsequently shown that these LST genes relate to a function of SEC13 in the sorting of amino acid permeases in the late secretory pathway, and analysis of these genes is described elsewhere (Roberg et al., 1997a,b).GeneLST genesGeneSEC1327140Mutations in LST1 were inviable when combined with sec16, sec23, and sec31 mutations, and mutations in LST6 were inviable with sec16 and sec31 (Table III).GeneLST1Genesec16Genesec23Genesec31GeneLST6Genesec1627297Importantly, mutations in LST1 and LST6 did not show synthetic lethality in parallel crosses to mutations in SEC17 or SEC18, genes required for fusion of COPII vesicles.GeneLST1GeneLST6GeneSEC17GeneSEC1827469Given that synthetic-lethal interactions usually occur between mutations in genes involved in the same step of the secretory pathway, the tests for genetic interactions indicated that LST1, and probably also LST6, participate in vesicle budding from the ER.GeneLST1GeneLST6title_22773127731Lst1p Is Homologous to Sec24pGeneLst1pGeneSec24pparagraph2776127761The LST1 gene was isolated by its ability to restore sectoring to CKY426 (MATa sec13-1 ade2 ade3 leu2 ura3 [pKR4]), a strain that forms solid red, nonsectoring colonies because of the presence of the lst1-1 mutation.GeneLST1 geneGeneCKY426GeneMATa sec13-1 ade2Geneade3 leu2 ura3GenepKR4Genelst1-127980CKY426 was transformed with yeast genomic libraries.GeneCKY4262803334 colonies that regained the ability to form white sectors were identified among 97,000 Ura+ transformants.28144We expected this screen to yield plasmids carrying either SEC13 or LST1.GeneSEC13GeneLST128218About half of the complementing plasmids were shown to carry SEC13 by restriction site mapping and by the ability to complement the temperature sensitivity of sec13-1.GeneSEC13Genesec13-128389The restriction maps of the remaining rescuing plasmids show that they represent two unrelated chromosomal regions.28506The clones p21-31 and p77-2 were selected as representatives of each region.Genep21-31Genep77-228584The genomic sequence from p77-2 (a clone in the plambdaYES vector; Elledge et al., 1991) was inserted as an XhoI fragment into the integrating vector pRS306 to produce pKR20.Genep77-2GeneXhoIGenepKR2028761For chromosomal integration, pKR20 was linearized by digestion with HpaI and transformed into CUY564 (MATalpha ade2 ade3 leu2 ura3).GenepKR20GeneHpaIGeneCUY564GeneMATalpha ade2 ade3 leu2 ura328897The resulting strain was crossed to the lst1-1 mutant CKY426 (MATa lst1-1 sec13-1 ade2 ade3 leu2 ura3 [pKR4]).Genelst1-1 mutant CKY426GeneMATaGenelst1-1 sec13-1 ade2Geneade3 leu2 ura3GenepKR429010After sporulation and dissection, the integrated pKR20 was found to be completely linked to the LST1 locus: sectoring segregated 2:2 and all sectored colonies were Ura+, whereas all nonsectored colonies were Ura-.GenepKR20GeneLST1 locusGeneUra+GeneUra29227Thus, p77-2 carries the LST1 gene.Genep77-2GeneLST1 gene29262In parallel, the genomic sequence from p21-31 (a clone in the pCT3 vector; Thompson et al., 1993) was inserted as an EcoRI/HindIII fragment into pRS306 to produce pKR7.Genep21-31GeneEcoRIGeneHindIIIGenepRS306GenepKR729433pKR7 was integrated at its chromosomal locus after linearization with MscI and was then checked for linkage to lst1-1.GenepKR7GeneMscIGenelst1-129553Tetrad analysis showed that pKR7 was not linked to LST1 and we concluded that pKR7 carries an unlinked suppressor gene.GenepKR7GeneLST1GenepKR7paragraph2967029670The 3.5-kb insert of p77-2 was inserted into the XhoI site of the centromeric vector pRS316 to construct pKR17.Genep77-2GeneXhoI site29784The base sequence of this insert was determined and found to contain a single open reading frame encoding a protein of 929 amino acids.29922This sequence corresponds to the open reading frame YHR098c located on chromosome VIII (Saccharomyces Genome Database, Cherry et al., 1997).30066The predicted amino acid sequence of LST1 shows significant similarity to SEC24 (YIL109C).GeneLST1GeneSEC24GeneYIL109C30158The two proteins share 23% sequence identity that extends over most of their length (Fig. 2), suggesting that Lst1p may have a function similar to that of Sec24p as a subunit of the COPII vesicle coat.GeneLst1pGeneSec24pGeneCOPII vesicle coattitle_23036430364Phenotypes of lst1DeltaGenelst1Deltaparagraph3038530385One copy of the LST1 gene in the wild-type diploid strain CKY348 was disrupted to generate a lst1Delta::LEU2/LST1 heterozygote.GeneLST1 geneGeneLEU2GeneLST130515Sporulation and dissection of this diploid gave >95% spore viability on YPD medium and the LEU2 marker segregated 2:2, showing that LST1 is not essential for growth.GeneLEU2GeneLST130683A lst1Delta::LEU2 mutant spore clone was crossed to sec mutants to test for synthetic lethality.GeneLEU2 mutant spore clone30781In these crosses, both the temperature sensitivity of the sec mutation and the lst1Delta allele marked by LEU2 could be followed independently.Genelst1Delta alleleGeneLEU230928In crosses of lst1Delta to sec12, sec13, sec16, sec23, sec24, or sec31 mutants, inviability segregated as a two-gene trait (segregation patterns for dead:viable spore clones were 2:2, 1:3, and 0:4).Genelst1DeltaGenesec12Genesec13Genesec16Genesec23Genesec24Genesec31 mutants31130Tests of the genotype of the surviving sister spore clones showed that the inviable spores in these crosses were always lst1Delta sec double mutants.Genelst1Delta sec double mutants31282Crosses between lst1Delta and sec17 or sec18 produced viable double mutants.Genelst1DeltaGenesec17Genesec1831359These findings confirmed and extended our earlier tests for synthetic lethality with lst1-1, and demonstrated that lst1Delta was synthetically lethal with all the known genes required for COPII vesicle formation, but not with genes required for vesicle fusion.Genelst1-1Genelst1Deltaparagraph3160231602We evaluated the growth of lst1Delta::LEU2 mutants under a variety of conditions.Genelst1DeltaGeneLEU2 mutants31685On YPD, the lst1Delta::LEU2 strain grew, as well as an isogenic wild-type strain at temperatures ranging from 14 to 37 C.Genelst1DeltaGeneLEU231808However, on SMM the lst1Delta::LEU2 strain grew poorly at temperatures above 30 C.GeneLEU231893Since YPD (pH 6.5) and SMM (pH 3.8) differed markedly in pH, we suspected that lst1Delta mutants may be particularly sensitive to an acidic environment, and we tested the effect of pH on the growth of lst1Delta mutants.Genelst1Delta mutantsGenelst1Delta mutants32116Although lst1Delta mutants grew as well as wild-type on YPD at all temperatures, when YPD was brought to pH 3.8, lst1Delta mutants grew much more slowly than wild-type at 37 C (Fig. 3 A).Genelst1Delta mutantsGenepH 3.8Genelst1Delta mutants32307Conversely, on SMM buffered to pH 6.5, lst1Delta and wild-type grew even at 37 C (data not shown).32407These results demonstrated that at high temperature, growth of the lst1Delta mutant was sensitive to acidic conditions.Genelst1Delta mutantparagraph3250732507Having identified conditions where LST1 was needed for growth, we investigated whether overexpression of SEC24 could supply the function lost in lst1Delta.GeneLST1GeneSEC2432665Some restoration of function was indicated by the ability of an lst1Delta mutant to grow on acidic medium when provided with extra copies of SEC24 on either centromeric or 2mu plasmids (Fig. 3 B).Genelst1Delta mutantGeneSEC24GeneFig. 3 B32864These findings imply some functional overlap between LST1 and SEC24.GeneLST1GeneSEC2432934In parallel tests for suppression, we found that the genes SEC12, SEC13, SEC31, or SEC23, when expressed from 2mu plasmids, could not restore the ability of an lst1Delta mutant to grow on acidic medium.GeneSEC12GeneSEC13GeneSEC31GeneSEC23Genelst1Delta mutant33138We found that the lst1Delta mutation caused a selective defect in the trafficking of Pma1p from the ER, and we also examined the ability of overexpressed SEC24 to suppress this phenotype caused by the lst1Delta mutation.Genelst1DeltaGenePma1pGeneSEC24Genelst1Delta33361By immunofluorescence microscopy, the proper localization of Pma1p to the cell surface was restored in an lst1Delta strain that also carried SEC24 on a 2mu plasmid (see Fig. 5).GenePma1pGenelst1DeltaGeneSEC24GeneFig. 5paragraph3352433524In an attempt to test the effect of overexpression of LST1, we found that LST1 on a 2mu plasmid severely impaired growth of wild-type yeast cells.GeneLST1GeneLST133672To examine the response of cells to different doses of Lst1p, we designed a way to express different levels of Lst1p according to the amount of galactose in the growth medium.GeneLst1pGeneLst1p33850A wild-type strain (CKY473) carrying a plasmid that expressed LST1 from pGAL1 (pKR35) was spread on an SMM plate with 2% raffinose, a carbon source that allows yeast growth without repression of the GAL1 promoter.GeneLST1GenepGAL1GenepKR35GeneGAL1 promoter34068When these cells are exposed to a gradient of galactose concentrations, from 3 mg of galactose in a filter disk on top of the lawn, growth was inhibited in a halo 1.5 cm beyond the edge of the filter (Fig. 3 C).34284A strain that did not contain pKR35 grew uniformly up to the edge of the filter, showing that the galactose itself was not inhibitory.GenepKR3534421Given the similarity of Lst1p to Sec24p, we asked whether the overexpression of SEC24 could compensate for overexpression of LST1.GeneLst1pGeneSec24pGeneSEC24GeneLST134555Cells carrying both the pGAL1-LST1 plasmid (pKR35) and the SEC24 gene on a 2mu plasmid (pKR41) were tested in an identical halo assay, and were found to be resistant to the effect of galactose (Fig. 3 C).GenepGAL1-LST1 plasmidGenepKR35GeneSEC24 geneGenepKR4134763Suppression by SEC24 appeared to be specific, since parallel tests of 2mu plasmids carrying SEC12, SEC13, SEC31, or SEC23 failed to show suppression.GeneSEC24GeneSEC12GeneSEC13GeneSEC31GeneSEC2334916It is worth noting that SEC23 expressed from a 2mu plasmid significantly slows the growth of our yeast strains.GeneSEC2335030Any suppression afforded by overexpression of SEC23 might be counteracted by this inherent toxicity of SEC23.GeneSEC23GeneSEC2335142A simple conclusion that can be drawn from these overexpression studies is that too great of a stoichiometric excess of Lst1p over Sec24p is lethal.GeneLst1p35293This observation can be explained if Lst1p and Sec24p compete with one another in the assembly of vesicle coat complexes and that excess Lst1p causes sequestration of vesicle components into complexes that fail to satisfy some essential function of COPII.GeneLst1pGeneSec24pGenevesicle coat complexesGeneLst1ptitle_23555535555lst1Delta Diminishes the Activity of the Plasma Membrane Proton-ATPaseGenelst1DeltaGenePlasma Membrane Proton-ATPaseparagraph3562335623The sensitivity of lst1Delta mutants to low pH suggested the involvement of Pma1p, which has been shown to be the limiting cell component for growth on acidic medium (McCusker et al., 1987; Portillo and Serrano, 1989).Genelst1Delta mutantsGenePma1p35844The dependence of Pma1p activity on LST1 was supported by the observation that lst1Delta mutants exhibited an unusual morphology characteristic of pma1 mutants.GenePma1pGeneLST1Genelst1Delta mutantsGenepma1 mutants36006When lst1Delta mutants were grown in low pH (SMM or YPD brought to pH 3.8) at 37 C, ~10% of the cells formed multibudded rosettes; in some cases, as many as 15 daughters radiated from a single large mother cell (Fig. 4 A).Genelst1Delta mutants36231The unseparated daughter cells contained nuclei that could be stained with DAPI and the daughter cells could be separated from their mothers by micromanipulation, indicating they had completed cytokinesis.36441Cells depleted of Pma1p produce similar multibudded cells with attached daughters that had completed cytokinesis.GenePma1p36557In this case, multibudded rosettes are thought to form because a mother cell formed with sufficient Pma1p in the plasma membrane will continue to bud, whereas daughter cells formed after Pma1p transport is compromised will have insufficient Pma1p to form buds themselves (Cid et al., 1987).GenePma1pGenePma1p transportGenePma1p36853The morphology of lst1Delta cells grown at relatively high pH (YPD or SMM buffered to pH 6.5) at 37 C appeared normal, with few cells having more than one attached daughter.paragraph3702337023As a more direct test of the effect of lst1Delta on the activity of Pma1p, we measured the capacity of mutant cells to pump protons into the external medium.Genelst1DeltaGenePma1p37183Wild-type and lst1Delta strains were cultured in YPD at 37 C, conditions under which both strains grow equally well.37301After starvation by prolonged incubation in water, the cells were placed in a weakly buffered medium and proton efflux on addition of glucose was measured as a drop in extracellular pH.37490For both wild-type and lst1Delta strains, addition of glucose produced a sharp decline in pH (after a 30-s lag), which began to level off after ~5 min (Fig. 4 B).GeneFig. 4 B37655Although the responses of wild-type and lst1Delta cells were qualitatively similar, proton efflux from lst1Delta cells was compromised: in the first 5 min after addition of glucose the rate of change in pH produced by the lst1Delta mutant was 65% of that of wild-type.37928These findings indicate that the lst1Delta mutant grown at 37 C has about half of the Pma1p activity as wild-type cells.Genelst1Delta mutantGenePma1ptitle_23803338033LST1 Is Required for Efficient Transport of Pma1p Out of the ERGeneLST1GenePma1p Outparagraph3809738097To determine whether the reduced Pma1p activity in lst1Delta mutants was due to a defect in the transport of Pma1p to the cell surface, we compared the localization of Pma1p in wild-type and lst1Delta mutant cells by immunofluorescence microscopy.GenePma1pGenelst1Delta mutantsGenePma1pGenePma1p38349Cells were grown at 30 C in YPD medium to avoid possible secondary effects due to the pH sensitivity of lst1Delta mutants.Genelst1Delta mutants38474In lst1Delta cells, Pma1p was located primarily at the nuclear periphery and at the cellular rim, indicating that a large proportion of Pma1p remains in the ER (Fig. 5).GenePma1pGenePma1p38645This pattern of localization differed markedly from the surface localization of Pma1p in wild-type cells incubated at 30 C (Fig. 5) or in lst1Delta cells incubated at 24 C (data not shown).GenePma1pparagraph3882638826We also examined the subcellular distribution of Pma1p in lst1Delta cells by cell fractionation.GenePma1p38924Lysates from cells grown at 37 C for 3 h were fractionated on sucrose density gradients under conditions where the ER and plasma membrane are well separated on the basis of their buoyant density.39121Pma1p from wild-type cells was located in dense fractions of the gradient in a peak that was coincident with that of Gas1p, a GPI-linked plasma membrane protein (Nuoffer et al., 1991).GenePma1pGeneGas1p39309In contrast, <35% of the total Pma1p from lst1Delta cells coincided with the plasma membrane marked by Gas1p protein and the majority of Pma1p was located in fractions containing the ER (Fig. 6).GenePma1pGeneGas1p proteinGenePma1p39508Interestingly, the ER from lst1Delta mutants (marked by Sec61p) reproducibly resolved into two peaks of different density, suggesting that accumulation of Pma1p segregates ER membranes into subdomains of relatively high and low density.GeneER from lst1Delta mutantsGeneSec61pGenePma1p39749Given that most of the Pma1p was located in the ER peak of higher density, it is possible that the density of the ER had been increased because of the accumulation of Pma1p.GenePma1pGenePma1p39926A similar increase in density of a portion of the ER is caused when folding mutants of PMA1 are retained within the ER (Harris et al., 1994).GeneERGenePMA1paragraph4006340063The fact that transport of Pma1p, but not of Gas1p, was affected by deletion of LST1 suggested that LST1 may be specifically required for the export of Pma1p from the ER.GenePma1pGeneGas1pGeneLST1GeneLST1GenePma1p40237The absence of a general protein secretion defect in lst1Delta mutants was implied by the normal growth of lst1Delta mutants at 37 C in medium of pH 6.5 (the doubling time of both lst1Delta and wild-type was 1.75 h in YPD), indicating a normal rate of expansion of the plasma membrane.Genelst1Delta mutantsGenelst1Delta mutantsGenelst1Delta40526As a specific test for the rate of ER to Golgi transport, pulse- chase experiments were performed to follow the rate of maturation of invertase from its core glycosylated ER form to the Golgi and secreted forms.Geneinvertase40741No delay in invertase transport was observed in lst1Delta mutants that had been grown at 37 C for 3 h, conditions that caused the accumulation of Pma1p (Fig. 7).Geneinvertase40904Similarly, no defect in the maturation of carboxypeptidase Y from the ER form to the Golgi and vacuolar forms of the enzyme could be detected (data not shown).Genecarboxypeptidase Yparagraph4105941059We also considered the possibility that transport of Pma1p may be particularly sensitive to any subtle defect in vesicle formation.GenePma1p41193We addressed this possibility by examining the localization of Pma1p in sec24-1 and sec31-2 mutant cells at the semipermissive temperature of 28 C.GenePma1pGenesec24-1Genesec31-241341Although the growth rate of both mutants was compromised at this temperature (doubling time on YPD: 2.9 h for sec24 and 2.4 h for sec31, as compared with 1.7 h for wild-type), no accumulation of Pma1p was detected in the perinuclear region of either mutant by immunofluorescence microscopy (data not shown).GenePma1p41653Thus, partial defects in COPII functions did not lead to the extensive accumulation of Pma1p in the ER that was observed for lst1Delta mutants.GenePma1pGenelst1Delta mutants41800Taken together, comparisons between the lst1Delta mutation and COPII gene mutations indicate that the lst1Delta mutation is unusual in its ability to inhibit Pma1p exit from the ER without interfering with the transport of other cargo proteins.GeneCOPII geneGenelst1DeltaGenePma1ptitle_24204042040Localization of Lst1pGeneLst1pparagraph4206242062To examine the intracellular distribution of Lst1p, an epitope-tagged derivative was constructed by inserting six copies of the 10-amino acid HA near the NH2 terminus of Lst1p.GeneLst1pGeneLst1p42242The HA-tagged LST1 was functional, as demonstrated by its ability to complement lst1-1 in a sectoring assay, and to restore the ability of a lst1Delta mutant to grow on acidic medium at 37 C (not shown).GeneLST1Genecomplement lst1-1Genelst1Delta mutant42447In cells expressing Lst1p-HA that were fixed for immunofluorescence microscopy, staining was found primarily at the nuclear periphery (Fig. 8).GeneLst1p-HA42592No signal was seen in cells expressing untagged Lst1p, verifying that the origin of the staining pattern was due to Lst1p-HA.GeneLst1pGeneLst1p-HA42719Although Lst1p-HA staining largely coincided with the ER marker Kar2p, there were subtle differences in their patterns of localization: Kar2p appeared uniformly, distributed around the nuclear periphery, whereas Lst1p-HA staining had a more punctate appearance indicating that Lst1p might be concentrated in particular regions of the ER.GeneLst1p-HAGeneER marker Kar2pGeneKar2pGeneLst1p-HAGeneLst1p43060In addition, weak punctate staining was observed throughout the cell body, some of which may correspond to ER membranes near the cell periphery.GeneERparagraph4320943209The intracellular distribution of Lst1p was also examined by subcellular fractionation.GeneLst1p43297Cells expressing Lst1p-HA were converted to spheroplasts and then gently lysed.GeneLst1p-HA43378This cell lysate was subjected to differential centrifugation and most of Lst1p-HA was found to pellet at either 500 g or 10,000 g (Fig. 9 A).GeneLst1p-HA43523All of the soluble marker protein Gdh2p (Miller and Magasanik, 1990) was found in the 150,000 g supernatant fraction, indicating complete cell lysis (data not shown).43692The association of the Lst1p protein with the sedimenting fraction was analyzed by chemical treatment of cell lysates before centrifugation at 50,000 g.GeneLst1p protein43848Incubation of cell extracts in 1% Triton X-100, 2.5 M urea, 100 mM sodium carbonate, pH 11.5, or 500 mM NaCl resulted in the release of a portion of the Lst1p-HA into the soluble fraction (Fig. 9 B).GeneLst1p-HAGeneFig. 9 B44050The partial dissociation of Lst1p-HA from the sedimenting fraction by these agents suggested that Lst1p is a peripheral membrane protein that adheres tightly to the membrane.GeneLst1p-HAGeneLst1ptitle_24422944229Lst1p Binds Sec23pGeneLst1pGeneSec23pparagraph4424844248Sec24p was first identified as a protein that formed a 400-kD complex with Sec23p (Hicke et al., 1992).GeneSec24pGeneSec23p44353Because of the similarity of Lst1p to Sec24p, we investigated whether Lst1p could also bind to Sec23p.GeneLst1pGeneSec24pGeneLst1pGeneSec23p44458To assay potential interactions by the yeast two-hybrid assay, LST1 was fused to the lexA DNA-binding domain (pKR37) and SEC23 was fused to an acidic activation domain (pPE81).GeneLST1GenelexA DNA-binding domainGenepKR37GeneSEC2344637Interaction between the two fusion proteins was tested by assaying for activation of a lacZ reporter gene.GenelacZ reporter gene44746Induction of beta-galactosidase was observed when the LST1 and SEC23 fusions were coexpressed, but not when expressed alone (Table IV).Genebeta-galactosidaseGeneLST1GeneSEC2344884The level of induction caused by interaction of LST1 and SEC23 was similar to that seen for interaction of SEC24 and SEC23 (Gimeno et al., 1996).GeneLST1GeneSEC23GeneSEC24GeneSEC23paragraph4503145031To confirm the interaction between Lst1p and Sec23p, association of these proteins was examined in yeast cell extracts.GeneLst1pGeneSec23p45152The coding sequence of LST-1-HA (codons 14-927) was fused to GST and expressed in yeast from the pGAL1 promoter.GeneLST-1-HAGeneGSTGenepGAL1 promoter45267SEC23 was also expressed from pGAL1.GeneSEC23GenepGAL145304Since both proteins are largely associated with intracellular membranes (Fig. 10 B), membranes prepared from cells overexpressing both Sec23p and GST-Lst1p-HA were first extracted with 600 mM NaCl to release protein complexes from the membrane, the salt extracts were clarified by centrifugation at 90,000 g, and diluted to give a final concentration of 200 mM NaCl.GeneSec23pGeneGSTGeneLst1p45677GST-Lst1p-HA was isolated from the extracts by affinity to glutathione Sepharose beads.GeneGSTGeneLst1p45767Sec23p was found in association with GST-Lst1p-Ha, but not in control extracts prepared from cells expressing Sec23p and GST alone (Fig. 10 A).GeneSec23pGeneGSTGeneLst1pGeneHaGeneSec23pGeneGST45913Together, these experiments show that Lst1p, like Sec24p, can form a complex with Sec23p.GeneLst1pGeneSec24pGeneSec23pparagraph4601346013Sec23p and Sec24p have been shown to assemble onto the ER membrane as a complex (Matsuoka et al., 1998).GeneSec23pGeneSec24p46120While working out conditions to optimize recovery of Sec23p bound to GST-Lst1p-HA, we discovered that assembly of an Lst1p/Sec23p complex appears to enhance the association of both proteins with the ER membrane.GeneSec23pGeneGSTGeneLst1pGeneHAGeneLst1pGeneSec23p46335When both GST-Lst1p-HA and Sec23p were overexpressed in the same cell, >60% of the Sec23p, and 70% of the GST-Lst1p-HA were found in a fraction that pelleted at 10,000 g (Fig. 10 B).GeneGSTGeneLst1pGeneHAGeneSec23pGeneSec23pGeneGSTGeneLst1pGeneHA46520This pellet contains most of the ER, as marked by the ER membrane protein Sec61p (data not shown).GeneER membrane protein Sec61p46621When material that pelleted at 10,000 g was suspended in 60% sucrose and applied to the bottom of a sucrose density gradient, >90% of the GST- Lst1p-HA and Sec23p cofractionated with the ER resident membrane protein, Sec61p, at a density corresponding to 45% sucrose, showing that GST-Lst1p-HA and Sec23p were associated with membranes (data not shown).GeneGSTGeneLst1pGeneHAGeneSec23pGeneER resident membrane proteinGeneSec61pGeneGSTGeneLst1pGeneHAGeneSec23p46980In contrast to the case when Sec23p and GST-Lst1p-HA were expressed together, <10% of the Sec23p pelleted at 10,000 g in lysates from a strain overexpressing Sec23p alone.GeneSec23pGeneGSTGeneLst1pGeneHAGeneSec23pGeneSec23p47154Similarly, <20% of the GST-Lst1p-HA pelleted at 10,000 g in lysates from a strain expressing GST-Lst1p-HA alone (Fig. 10 B).GeneGSTGeneLst1pGeneHAGeneGSTGeneLst1p47281Thus, when either Sec23p or GST-Lst1p-HA was overexpressed alone, most of the overexpressed protein was soluble, but when the proteins were expressed together, most of the proteins were associated with the ER membranes.GeneSec23pGeneGSTGeneLst1pGeneHA47503These data support the observation that Lst1p can form a complex with Sec23p, and that the Lst1p/ Sec23p complex has affinity for ER membranes.GeneLst1pGeneSec23pGeneLst1pGeneSec23ptitle_14766647666Discussionparagraph4767747677By screening for mutants that exhibited synthetic-lethal genetic interactions with the COPII mutation sec13-1, we identified the LST1 gene.GeneCOPII mutation sec13-1GeneLST1 gene47819Subsequent genetic tests showed that lst1Delta is lethal when combined with mutations in genes required for COPII vesicle budding from the ER (SEC12, SEC13, SEC16, SEC23, SEC24, and SEC31), but lst1Delta is not lethal when combined with mutations in genes that are required for vesicle fusion with the Golgi compartment (SEC17 and SEC18).Genelst1DeltaGeneERGeneSEC12GeneSEC13GeneSEC16GeneSEC23GeneSEC24GeneSEC31GeneSEC17GeneSEC1848164This pattern of genetic interactions indicated that LST1 participates in the process of vesicle budding from the ER, an expectation that was born out by the examination of the LST1 gene and its product.GeneLST1GeneLST1 gene48370The following observations indicate a role for Lst1p as part of a COPII-like vesicle coat: (I) LST1 encodes a 90-kD protein that is homologous to the COPII-coat subunit Sec24p.GeneLst1pGeneLST1GeneCOPII-coat subunit Sec24p48549The two proteins share 23% amino acid identity over their entire lengths.48624(II) Lst1p is a peripheral ER membrane protein as shown by immunofluorescence microscopy and cell fractionation.GeneLst1pGeneER membrane protein48738(III) Lst1p, like Sec24p, can bind to Sec23p as shown by tests for two-hybrid interaction and affinity purification of a complex of GST-Lst1p and Sec23p.GeneLst1pGeneSec24pGeneSec23pGeneGSTGeneLst1pGeneSec23p48894(IV) Assembly of the Sec23p-Lst1p complex appears to enhance the membrane association of both Lst1p and Sec23p: when both proteins are overexpressed together, most associate with membranes, whereas either protein overexpressed alone is mostly cytosolic.GeneSec23pGeneLst1pGeneLst1pGeneSec23p49152(V) Although strains with chromosomal deletion of LST1 are viable and appear normal for secretion of marker proteins, these mutants show a pronounced accumulation of Pma1p in the ER, indicating a selective defect in ER to Golgi traffic.GeneLST1GenePma1pGeneER49393Based on these findings, we propose that Lst1p takes the place of Sec24p in a specialized COPII coat complex that is used for the recruitment of Pma1p into vesicles.GeneLst1pGeneSec24pGeneCOPII coat complexGenePma1pparagraph4955949559Strains carrying lst1Delta have the phenotypic hallmarks of a deficiency in Pma1p activity, including sensitivity to growth in an acidic environment, the formation of multibudded cells, and a decreased rate of proton efflux from intact cells.Genelst1DeltaGenePma1p49805All three traits are expressed only at temperatures of 30 C and above, indicating that LST1 is only required for Pma1p activity at high temperature.GeneLST1GenePma1p49954Localization of Pma1p in lst1Delta cells by immunofluorescence and sucrose density cell fractionation demonstrate that the transport of Pma1p from the ER is compromised in lst1Delta at 37 C.GenePma1pGenePma1pGeneERparagraph5014150141Export of Pma1p from the ER cannot be completely dependent on Lst1p, since Pma1p transport appears normal in lst1Delta mutants at 24 C.GenePma1pGeneLst1pGenePma1p transport50278Even at 37 C, the block in Pma1p transport may not be complete since ~35% of the total Pma1p fractionates with the plasma membrane, although some of the Pma1p detected in the plasma membrane in this experiment was probably synthesized before the shift to restrictive temperature.GenePma1p transportGenePma1pGenePma1p50563Therefore, it seems likely that Lst1p and Sec24p share the burden of transporting Pma1p from the ER.GeneLst1pGeneSec24pGenePma1p50666At 24 C, it appears that Sec24p (or some other protein) can compensate for the absence of Lst1p, but at temperatures of 30 C or higher, compensation is no longer possible unless extra copies of Sec24p are provided by expression from a multicopy plasmid.GeneSec24pGeneLst1pGeneSec24pparagraph5092750927The transport defect caused by deletion of LST1 appears to be specific for Pma1p.GeneLST1GenePma1p51009Under conditions where a defect in Pma1p transport was observed in lst1Delta mutants, transport of Gas1p, carboxypeptidase Y, and invertase was unaffected.GenePma1p transportGenelst1Delta mutantsGeneGas1pGenecarboxypeptidase YGeneinvertase51168Using growth as a more general assay for trafficking defects, we found that lst1Delta mutants grew at an identical rate to wild-type at 37 C when we compensated for the defect in Pma1p transport by using media at pH 6.5.Genelst1Delta mutantsGenePma1p transport51393This indicates that rate of expansion of the plasma membrane, including the transport of all essential plasma membrane proteins, is not significantly affected by the absence of LST1.GeneLST1paragraph5157351573We also considered the possibility that there may be differences among cargo molecules in their response to general defects in the protein transport machinery.51733Of particular concern was the possibility that Pma1p transport might be particularly sensitive to slowed ER to Golgi transport, such that a defect in transport too subtle to have an effect on our standard marker proteins might have a significant effect on the rate of transport of Pma1p.GenePma1p transportGenePma1p52025If this were the case, partial defects in other COPII components should also interfere with Pma1p transport.GenePma1p transport52136Therefore, we examined sec24 and sec31 mutants, but could find no evidence for a defect in Pma1p transport, even at semipermissive temperatures where the rate of growth was inhibited.Genesec24Genesec31 mutantsGenePma1p transport52322Although Pma1p was the only essential protein for which we could detect a transport defect in lst1 mutants, a defect in the transport of any nonessential protein could have been overlooked by our analysis.GenePma1pGenelst1 mutantsparagraph5253152531Factors required for the transport of specific membrane proteins have been documented in a number of other cases.52647The SHR3 gene encodes an ER resident protein that is required for the transport of amino acid permeases out of the ER, but is not required for the transport of a variety of other proteins (Ljungdahl et al., 1992; Kuehn et al., 1996).GeneSHR3 geneGeneERGeneER52884A set of ER proteins, Vma12p, Vma21p, and Vma22p, are required for transport from the ER of the integral membrane subunit of the vacuolar ATPase (Hill and Stevens, 1994, 1995; Jackson and Stevens, 1997).GeneER proteinsGeneVma12pGeneVma21pGeneVma22pGenevacuolar ATPase53090Similarly, mutational studies have shown that the small ER membrane protein Erv14p is specifically required for transport of the plasma membrane protein Axl2p out of the ER (Powers and Barlowe, 1998).Genesmall ER membrane protein Erv14pGeneplasma membrane protein Axl2p53294Finally, Ast1p has been suggested to be a factor specifically needed for the transport of Pma1p from the Golgi compartment to the plasma membrane (Chang and Fink, 1995).GeneAst1pGenePma1p53467In all of these cases, the question remains whether Shr3p, the Vma proteins, Erv14p, or Ast1p act directly in vesicular transport of their respective cargo molecules, or whether they are primarily involved in protein folding and influence protein sorting indirectly through quality control mechanisms.GeneShr3pGeneVma proteinsGeneErv14pGeneAst1p53772Because Lst1p appears to be a component of a vesicle coat, Lst1p seems more likely to have a direct role in the sorting of Pma1p rather than in its folding.GeneLst1pGeneLst1pGenePma1pparagraph5393153931Expression of a variety of dominant PMA1 mutations can cause accumulation of both mutant and wild-type Pma1p in proliferated ER (Harris et al., 1994; Portillo, 1997).Genedominant PMA1 mutationsGenePma1p54101Similarly, the transport of wild-type Pma1p from the ER is blocked when PMA2 (an isoform of PMA1) or plant plasma membrane proton-ATPases are overexpressed in yeast (Villalba et al., 1992; Supply et al., 1994; de Kerchove d'Exaerde et al., 1995).GenePma1pGenePMA1GeneATPases54351One proposal was that a special factor may be required for the transport of Pma1p from the ER in a manner analogous to the requirement for Shr3p in the transport of amino acid permeases (Supply et al., 1994).GenePma1pGeneShr3p54563The specific role of Lst1p in the transport of Pma1p suggests that it may be the factor depleted by the expression of dominant forms of Pma1p.GeneLst1pGenePma1pGenePma1p54707In the future, it may be possible to test this idea by evaluating the ability of Lst1p overexpression to reverse the effects of dominant PMA1 mutations.GeneLst1pparagraph5486354863The mechanism by which Lst1p acts in the transport of Pma1p may be inferred from recent studies examining the recruitment of cargo molecules into COPII vesicles.GeneLst1pGenePma1p55027Using ER-derived microsomes and purified COPII components, Kuehn et al. (1998) have shown that the Sec23p/Sec24p complex, along with Sar1p, associate with amino acid permeases and other integral membrane protein that are destined for the plasma membrane.GeneSec23pGeneSec24pGeneSar1p55285In parallel experiments using mammalian microsomes, mammalian Sec23p/Sec24p and Sar1p were found to bind to microsomal membranes and form a complex that contains the cargo protein VSV-G (Aridor et al., 1998).GeneSec23pGeneSec24pGeneSar1p55498The conclusion from both experimental systems is that the Sec23p/Sec24p complex contains specific binding sites for the capture of membrane cargo proteins within the plane of the ER membrane.GeneSec23pGeneSec24p55692Based on the data presented here, Lst1p appears to be an isoform of Sec24p that is adapted for selection of Pma1p.GeneLst1pGeneSec24pGenePma1p55809This provides the first evidence that Sec24 family members carry information specifying the type of cargo molecules that are accepted by ER-derived vesicles.GeneSec24 familyparagraph5596755967We have looked for association of Lst1p with ER-derived vesicles, but under the conditions of an in vitro budding reaction, a large quantity of Lst1p-HA is released from the membrane in soluble form.GeneLst1pGeneLst1p-HA56169Soluble Lst1p-HA gives a high background in vesicle fractions preventing us from reliably determining whether there is a specific association of Lst1p with vesicles.GeneLst1p-HAGeneLst1p56337In future experiments, it may be possible to isolate vesicles coated with Lst1p by performing an in vitro budding reaction using purified cytosolic components, including a purified complex of Lst1p and Sec23p.GeneLst1pGeneLst1pGeneSec23p56550It may also be possible to determine whether vesicles that are formed using a Sec23p/Lst1p complex more efficiently incorporate Pma1p than vesicles formed using the Sec23p/Sec24p complex.GeneSec23pGeneLst1pGenePma1pGeneSec23pGeneSec24p56741Finally, it will be of interest to determine if there is direct binding of Lst1p to Pma1p.GeneLst1pGenePma1pparagraph5683356833The identification of a Sec24p homologue that also acts in transport from the ER raises the possibility that the coats of ER-derived vesicles may be heterogeneous.GeneSec24p56999It is possible that Sec23p/Lst1p complexes act to form a class of vesicle that is distinct from those formed by Sec23p/ Sec24p complexes.GeneSec23pGeneLst1pGeneSec23pGeneSec24p57139Alternatively, it is possible that the two complexes assemble together forming vesicles with coats of mixed composition.57262The identification of additional homologues of Sec23p and Sec24p suggest the existence of coats with even greater combinatorial complexity.GeneSec23pGeneSec24p57403We have identified a third Sec24p family member, which we call Iss1p, as a protein that binds to Sec16p.GeneSec24p family memberGeneIss1pGeneSec16p57510Iss1p (YNL049c) also binds Sec23p and appears to be associated with the ER membrane (Gimeno, 1996).GeneIss1pGeneYNL049cGeneSec23p57612In addition, the Saccharomyces genome contains an uncharacterized open reading frame (YHR035w) that is 21% identical to Sec23p (Saccharomyces Genome Database, Cherry et al., 1997).57796If each of the Sec23p and Sec24p homologues carry different determinants for cargo selection, and if mixed coats can form, the possible combinations of Sec23p and Sec24p homologues should allow the formation of a wide variety of COPII-like vesicles with different capacities to carry different cargo molecules.GeneSec23pGeneSec24pGeneSec23pGeneSec24ptitle_15811158111Figures and Tablestable_caption5813058130S. cerevisiae Strainstable5815358156Strain Genotype Source or reference CUY563 MATaade2-101 ade3-24 leu2-3,112 ura3-52 T.GeneCUY563 MATaade2-101 ade3-24 leu2-358248Huffaker (Cornell University) CUY564 MATalpha ade2-101 ade3-2 leu2-3,112 ura3-52 T.GeneMATalpha ade2-101 ade3-2 leu2-358335Huffaker (Cornell University) EGY40 MATalpha ura3-52 leu2 his3 trp1 Golemis and Brent, 1992 CKY45 MATalpha sec13-1 his4-619 ura3-52 Kaiser Lab Collection CKY50 MATalpha sec16-2 his4-619 ura3-52 Kaiser Lab Collection CKY54 MATalpha sec17-1 his4-619 ura3-52 Kaiser Lab Collection CKY58 MATalpha sec18-1 his4-619 ura3-52 Kaiser Lab Collection CKY78 MATalpha sec23-1 his4-619 ura3-52 Kaiser Lab Collection CKY348 MATa/alpha leu2-3/leu2-3 ura3-52/ura3-52 Kaiser Lab Collection CKY423 MATalpha sec13-1 ade2-101 ade3-24 leu2-3,112 ura3-52 [pKR4] CKY424 MATasec13-1 ade2-101 ade3-24 leu2-3,112 ura3-52 [pKR4] CKY426 MATalst1-1sec13-1 ade2-101 ade3-24 leu2-3,112 ura3-52 [pKR4] CKY435 MATalst1-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY436 MATalst2-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY437 MATalst3-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY438 MATalst4-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY439 MATalst5-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY440 MATalst6-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY441 MATalst7-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY442 MATalst8-1 sec13-1::[SEC13, URA3] ade2-101 ade3-24 leu2-3,112 ura3-52 CKY443 MATa prototroph Kaiser Lab Collection CKY473 MATaleu2-3,112 ura3-52 Gal+ Kaiser Lab Collection CKY534 MATalpha lst1Delta::LEU2 leu2-3,112 ura3-52 [pKR17HA] CKY535 MATalst1Delta::LEU2 leu2-3,112 ura3-52 [pKR17HA] CKY536 MATalst1Delta::LEU2 ura3-52 leu2-3,112 CKY540 MATaleu2-3,112 ura3-52 [pNV31] CKY541 MATasec12-4 ura3-52 [pNV31] CKY542 MATalst1Delta::LEU2 leu2-3,112 ura3-52 [pNV31] CKY552 MATalpha lst1Delta::LEU2 leu2-3,112 ura3-52GeneMATalpha ura3-52 leu2 his3 trp1 Golemis and Brent, 1992 CKY45 MATalpha sec13-1 his4-619 ura3-52 Kaiser Lab Collection CKY50 MATalpha sec16-2 his4-619 ura3-52 Kaiser LabGeneCKY54 MATalpha sec17-1 his4-619 ura3-52 Kaiser LabGeneCKY58 MATalpha sec18-1 his4-619 ura3-52 Kaiser Lab Collection CKY78 MATalpha sec23-1 his4-619 ura3-52 Kaiser LabGeneCKY348 MATa/alpha leu2-3Geneleu2-3 ura3-52Geneura3-52 Kaiser Lab Collection CKY423 MATalpha sec13-1 ade2-101 ade3-24 leu2-3,112 ura3-52 [pKR4] CKY424 MATasec13-1 ade2-101 ade3-24 leu2-3,112 ura3-52GenepKR4GeneCKY426 MATalst1-1sec13-1 ade2-101 ade3-24 leu2-3,112 ura3-52GenepKR4GeneCKY435 MATalst1-1 sec13-1GeneSEC13GeneURA3Geneade2-101 ade3-24 leu2-3Gene112Geneura3-52 CKY436 MATalst2-1 sec13-1GeneSEC13GeneURA3Geneade2-101 ade3-24 leu2-3Gene112Geneura3-52 CKY437 MATalst3-1 sec13-1GeneSEC13GeneURA3Geneade2-101 ade3-24 leu2-3Gene112Geneura3-52 CKY438 MATalst4-1 sec13-1GeneSEC13GeneURA3Geneade2-101 ade3-24 leu2-3Gene112Geneura3-52 CKY439 MATalst5-1 sec13-1GeneSEC13GeneURA3Geneade2-101 ade3-24 leu2-3Gene112Geneura3-52 CKY440 MATalst6-1 sec13-1GeneSEC13GeneURA3Geneade2-101 ade3-24 leu2-3Gene112Geneura3-52 CKY441 MATalst7-1 sec13-1GeneSEC13GeneURA3Geneade2-101 ade3-24 leu2-3Gene112Geneura3-52 CKY442 MATalst8-1 sec13-1GeneSEC13GeneURA3Geneade2-101 ade3-24 leu2-3Gene112Geneura3-52 CKY443 MATa prototroph Kaiser Lab Collection CKY473 MATaleu2-3,112 ura3-52 Gal+ Kaiser Lab Collection CKY534 MATalpha lst1DeltaGeneLEU2 leu2-3,112 ura3-52 [pKR17HA] CKY535 MATalst1DeltaGeneLEU2 leu2-3,112 ura3-52 [pKR17HA] CKY536 MATalst1DeltaGeneLEU2 ura3-52 leu2-3,112 CKY540 MATaleu2-3,112 ura3-52 [pNV31] CKY541 MATasec12-4 ura3-52 [pNV31] CKY542 MATalst1DeltaGeneLEU2 leu2-3,112 ura3-52 [pNV31] CKY552 MATalpha lst1DeltaGeneLEU2 leu2-3Gene112Geneura3-52table_footnote6011460114All strains are from this study unless otherwise indicated.fig_caption6018060180Colony-sectoring screen for mutations that are lethal with sec13-1.Genesec13-160249CKY423 (ade2 ade3 leu2 ura3 sec13-1 [pKR4: SEC13, ADE3]) can lose the plasmid pKR4 when grown at 24 C on YPD, to give ade2 ade3 segregants that form white sectors within a red colony.GeneCKY423Geneade2 ade3 leu2 ura3 sec13-1GenepKR4GeneSEC13GeneADE3Geneade2 ade3 segregants60436Mutagenized cells that have acquired an lst mutation cannot grow without the pKR4 plasmid and form nonsectoring, solid red colonies.60570Of 132 nonsectoring colonies, the sectoring in 57 was restored by transformation with a second SEC13-bearing plasmid (pKR1).GeneSEC13-bearing plasmidGenepKR1table_caption6069860698Mutations Lethal with sec13-1Genesec13-1table6072960732Gene Number of alleles LST1 11 LST2 6 LST3 4 LST4 5 LST5 5 LST6 1 LST7 1 LST8 1 LST9 1 LST10 (SEC16) 2GeneLST1 11 LST2GeneLST3 4 LST4 5 LST5 5 LST6 1 LST7 1 LST8 1 LST9 1 LST10GeneSEC16table_caption6087560875Growth of lst sec Double Mutants at 24 Ctable6091860923sec13-1 sec16-2 sec23-1 sec31-1 sec17-1 sec18-1 lst1-1 - - - - + + lst2-1 - + + + ND ND lst3-1 - + + + ND ND lst4-1 +/- + + + ND ND lst5-1 - + + + ND ND lst6-1 - +/- - + - + + lst7-1 - + + + ND ND lst8-1 - + + + ND NDGenesec13-1 sec16-2 sec23-1 sec31-1 sec17-1 sec18-1 lst1-1 - - - - +Genelst2-1 - + + + NDGenelst3-1 - + + + ND ND lst4-1 +/- + + + NDGenelst5-1 - + + + ND ND lst6-1 - +/- - + - +Genelst7-1 - + + + ND ND lst8-1 - + + + ND NDtable_footnote6123161231Growth is represented in decreasing order by: + > +/- > +/-- > -.61297ND, not determined.fig_caption6133961339Comparison of LST1 and SEC24 sequences.GeneLST1GeneSEC2461380Identities are indicated by solid lines and similarities are indicated by dotted lines.61470Overall amino acid identity is 23%.fig_caption6150761507Functional relationships between LST1 and SEC24.GeneLST1GeneSEC2461557(A) Sensitivity of lst1Delta mutants to acidic medium.Genelst1Delta mutants61612Equal numbers of wild-type (CKY443) or lst1Delta::LEU2 (CKY534) cells were spotted onto YPD medium, pH 6.5, or acidic YPD medium (brought to pH 3.8 by the addition of HCl).GeneLEU2GeneCKY53461787Plates were photographed after incubation at 37 C for 2 d.61846(B) A lst1Delta::LEU2 strain (CKY552) was transformed with: vector only, pRS316; LST1 on a centromeric plasmid, pKR17; SEC24 on a centromeric plasmid, pAF70; or SEC24 on a 2mu plasmid, pKR34; and streaked onto YPD medium, pH 3.8.GeneLEU2GenepRS316GeneLST1GenepKR17GeneSEC24GenepAF70GeneSEC24GenepKR3462080Colonies were photographed after growth at 37 C for 2 d.62137(C) A wild-type strain (CKY473) was transformed either with a plasmid carrying pGAL1-LST1 (pKR35) and vector control (pRS425), or with pKR35 and SEC24 on a 2mu plasmid (pKR41).GenepGAL1GeneLST1GenepKR35GenepKR35GeneSEC24GenepKR4162317Transformants were plated at a density of 800 cells/cm2 on SMM plates containing 2% raffinose and then 3 mg galactose solution was placed on a sterile 1-cm filter on top of the lawn.62502The plates were photographed after growth at 30 C for 2 d.fig_caption6255762557Pma1p accumulates the ER in lst1Delta cells and this accumulation is suppressed by overexpression of SEC24.GenePma1pGeneSEC2462668Cells grown in SMM at 30 C were fixed with formaldehyde and then stained for immunofluorescence microscopy with affinity-purified anti-Pma1p antibody and FITC-conjugated secondary antibody.Geneanti-Pma1p antibody62862The same fields of cells, stained with DAPI to label the nuclear DNA, are also shown.62950Top panels, montage of lst1Delta cells (CKY536 carrying the empty vector pRS316); middle panels, genotypically wild-type cells (CKY536 carrying the LST1 plasmid pKR17); bottom panels, lst1Delta cells suppressed by SEC24 (CKY536 carrying the 2mu SEC24 plasmid pKR34).GeneCKY536GeneLST1 plasmid pKR17GeneSEC24GeneCKY536Gene2mu SEC24 plasmid pKR3463222Bar, 5 mum.fig_caption6322663226Pma1p defects caused by lst1Delta.GenePma1p63262(A) lst1Delta cells (CKY534) were photographed using differential interference contrast microscopy after growth at 37 C on YPD, pH 3.8.GeneCKY53463400A montage of multibudded cells is shown.63443Cells of this type comprise ~10% of a lst1Delta culture, but are never seen in wild-type grown under the same conditions.63568Bar, 10 mum.63581(B) Reduced capacity for proton pumping by lst1Delta cells.63642Wild-type (CKY443) and lst1Delta (CKY536) were grown to exponential phase in YPD medium, pH 6.8, at 37 C.Genelst1DeltaGeneCKY53663752Cells were incubated in water overnight and then suspended in 10 mM glycine buffer at pH 4.0.63847Proton efflux from the cells after addition of glucose was recorded as a decrease in the pH of the external medium.63963Based on the average rate of change in pH over the first 5 min after glucose addition, lst1Delta cells exhibited 65% the rate of proton efflux as wild-type.fig_caption6410864108Cell fraction to localize Pma1p in lst1Delta cells.GenePma1p64160Wild-type (CKY443) and lst1Delta (CKY536) cells were grown in YPD at 24 C and then were shifted to 37 C for 3 h.Genelst1DeltaGeneCKY53664275Cell lysates were fractionated on density gradients of 20-60% sucrose.64346Relative levels of Pma1p, Gas1p (plasma membrane marker), and Sec61p (ER marker) in each fraction were quantitated by immunoblotting and densitometry.GenePma1pGeneGas1pGeneSec61pGeneER64500GDPase (Golgi compartment marker) was determined by enzymatic assay.GeneGDPasefig_caption6456764567Transport of invertase is not affected by lst1Delta.Geneinvertase64620Wild-type (CKY540), lst1Delta (CKY542), and sec12-4 (CKY541) strains expressing invertase from the constitutive pTPI1-SUC2 fusion, were grown to exponential phase at 24 C in SMM medium, pH 6.5, without methionine.GeneWild-type (CKY540)Genelst1DeltaGeneCKY542Genesec12-4GeneCKY541GeneinvertaseGenepTPI1-SUC264837Wild-type and lst1Delta strains were shifted to 37 C, grown for 3 h, and the sec12-4 (CKY541) strain was shifted to 37 C 5 min before labeling.Genesec12-4GeneCKY54164983Cells were pulse-labeled with [35S]methionine and cysteine for 5 min and then chased by the addition of an excess of unlabeled methionine and cysteine.65136Invertase was immunoprecipitated from labeled extracts and resolved by SDS-PAGE.GeneInvertase65219Positions of the core glycosylated ER form and mature Golgi and secreted forms of invertase are indicated.Geneinvertasefig_caption6532265322Immunolocalization of Lst1p-HA.GeneLst1p-HA65354CKY535 (MATa lst1Delta::LEU2 leu2-3,112 ura3-52 [pKR17HA]) expressing Lst1p-HA from a centromeric plasmid was fixed and labeled with mouse anti-HA, FITC-conjugated anti-mouse antibodies, rabbit anti-Kar2p, and rhodamine-conjugated anti-rabbit antibodies.GeneCKY535GeneMATa lst1DeltaGeneLEU2 leu2-3,112 ura3-52GenepKR17HAGeneLst1p-HAGenemouse anti-HAGeneFITC-conjugated anti-mouse antibodiesGenerabbit anti-Kar2pGeneanti-rabbit antibodies65613Nuclear DNA was visualized by DAPI staining.65658Cell bodies were visualized by differential interference contrast microscopy (DIC).65744Bar, 1 mum.fig_caption6575765757The intracellular distribution of Lst1p.GeneLst1p65798(A) Cells expressing Lst1p-HA from a centromeric plasmid (CKY535) were gently lysed and subjected to sequential centrifugation steps, giving 500 g, 10,000 g, and 150,000 g pellet fractions (P) and a 150,000 g supernatant fraction (S).GeneLst1p-HAGeneCKY53566035Each sample contains extract from the same number of cells.66096(B) Cell lysates were treated for 1 h at 4 C with either 2.5 M urea, 500 mM NaCl, 100 mM sodium carbonate (pH 11.5), or 1% Triton X-100.66234Pellet (P) and supernatant (S) fractions were then separated by centrifugation at 50,000 g.66328Lst1p-HA was detected by SDS-PAGE and immunoblotting with anti-HA antibody.GeneLst1p-HAGeneanti-HA antibodytable_caption6640666406Two-Hybrid Interaction between LST1 and SEC23GeneLST1GeneSEC23table6645366458beta-galactosidase activity LST1 SEC24 No fusion SEC23 395 +- 8 629 +- 1 24.4 +- 0.1 No fusion 30.4 +- 4.0 25.3 +- 1.4 44.8 +- 5.3Genebeta-galactosidaseGeneLST1GeneSEC24table_footnote6659966599Fusions to the LexA DNA-binding domain and to a transcriptional activation domain were induced by growth in galactose for 10 h.GeneLexA DNA-binding domain66728Activities shown are the mean from five independent transformants.66796Units of beta-galactosidase activity are nmol/mg x min.Genebeta-galactosidasefig_caption6685966859Lst1p/-Sec23p complex is membrane associated.GeneLst1p/-Sec23p complex66905(A) Affinity isolation of Lst1p/-Sec23p complexes.GeneLst1p/-Sec23p complexes66957GST-Lst1p-HA or GST alone was coexpressed with Sec23p and isolated by affinity to glutathione Sepharose beads.GeneGSTGeneLst1pGeneHAGeneGSTGeneSec23p67069Proteins bound to glutathione beads were loaded in lanes 2 and 4.67135One-sixth of the total lysate was loaded in lanes 1 and 3.67194(B) GST-LST1-HA, SEC23, or both were expressed from the GAL1 promoter.GeneGSTGeneLST1GeneHAGeneSEC23GeneGAL1 promoter67266Cell lysates were cleared of cell debris by centrifugation at 300 g for 2 min.67347Pellet (P) and supernatant (S) fractions from cleared cell lysates were separated by centrifugation at 10,000 g for 30 min.67471An aliquot of the total cleared lysate (T) was removed before centrifugation.67550An equal number of cell equivalents were loaded for each sample.67616The GST-Lst1p-HA fusion was detected using anti-HA antibodies.GeneGSTGeneLst1pGeneHAGeneanti-HA antibodies67679For both A and B, the Sec23p protein was detected using anti-Sec23p antibodies.GeneSec23p proteinGeneanti-Sec23p antibodiestitle6776967769Abbreviations used in this paperparagraph6780267802DAPIparagraph67807678074',6-diamidino-2-phenylindoleparagraph6783967839GSTparagraph6784367843glutathione S-transferaseparagraph6786967869HAparagraph6787267872hemagglutinin epitopeGenehemagglutininparagraph6789467894LSTparagraph6789867898lethal with sec-thirteenparagraph6792467924Pma1pparagraph6793067930plasma membrane proton-ATPaseparagraph6796067960SMMparagraph6796467964supplemented minimal mediumparagraph6799267992YPDparagraph6799667996rich mediumtitle6800868008ReferencesProc Natl Acad Sci USAsurname:Hirschberg;given-names:CBsurname:Robbins;given-names:PWsurname:Orlean;given-names:P2476806surname:Abeijon;given-names:C861989ref6935693968019Topography of glycosylation in yeast: characterization of GDP mannose transport and lumenal guanosine diphosphatase activities in Golgi-like vesiclesJ Cell 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