LST1 Is a SEC24 Homologue Used for selective export of plasma membrane atpase from endoplasmic reticulum  In saccharomyces cerevisiae, vesicles that  carry protein from er to golgi compartment  are encapsulated by copii coat protein. We identified  mutation in gene, designated lst (lethal with sec-thirteen), that were lethal in combination with copii   mutation sec13-1. LST1 showed synthetic-lethal interaction with complete set of copii gene, indicating  that LST1 encodes a new COPII function. LST1 codes  for a protein similar in sequence to copii subunit  sec24p. Like Sec24p, lst1p is peripheral er membrane protein that binds to copii subunit sec23p.  chromosomal deletion of LST1 is not lethal, but inhibits  transport of plasma membrane proton-ATPase  (pma1p) to cell surface, causing poor growth on medium of low ph. localization by immunofluorescence   microscopy cell fractionation shows that export  of Pma1p from the ER is impaired in lst1delta mutant.  Transport of other proteins from the ER was not affected by lst1delta, nor was pma1p transport found to be  particularly sensitive to copii defect. Together,  finding suggest that specialized form of copii coat subunit, with Lst1p in place of Sec24p, is  used for efficient packaging of Pma1p into vesicle  derived from the ER. plasma membrane proton-atpase (Pma1p)1 is  essential integral membrane protein that couples  ATP hydrolysis to translocation of proton  across the plasma membrane (serrano et al., 1986). proton gradient generated by Pma1p then drives uptake of nutrient, such as amino acid, from extracellular medium (vallejo and Serrano, 1989). second physiological function of Pma1p is to maintain cytosol at neutral ph. In medium of low ph, growth rate is limited by the amount of cellular pma1p (McCusker et al.,  1987; portillo and Serrano, 1989). Pma1p transport to the  cell surface depends upon secretory pathway defined  by sec gene (brada and Schekman, 1988; Chang and  Slayman, 1991). Pma1p is one of abundant cargo   molecule of the secretory pathway, constituting 25-50  of total plasma membrane protein (Serrano, 1991). Because of abundance and physiological importance, one  might expect that yeast cell would have specialized mechanism to ensure efficient transport of Pma1p through the  secretory pathway. 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). For early step in secretory   pathway, proteins that are specifically required for the  transport of Pma1p have not yet been identified. Proteins destined for the plasma membrane are transported from the ER to the Golgi compartment by vesicles  coated with set of proteins known as copii barlowe et   al., 1994). copii coat are thought to cause deformation of membrane into a vesicle and to recruit  cargo molecule into vesicle bud (reviewed by Schekman  and orci, 1996). stepwise recruitment and assembly of  the COPII coat onto the membrane is thought to occur as  follows. action of er resident membrane protein  sec12p, guanine nucleotide exchange factor for sar1p,  causes Sar1p to bind to er membrane (barlowe and  schekman, 1993). membrane-associated sar1p, in turn, recruits soluble sec23p/sec24p and sec13p/sec31p complex (Matsuoka et al., 1998). sec16p resides on er   membrane and binds to sec23p/sec24p   sec13p/sec31p complex, likely organizing their assembly  onto the membrane (Espenshade et al., 1995; Gimeno et al.,  1996; Shaywitz et al., 1997). To examine role of different   copii coat subunit in recruitment of cargo molecules to  vesicles, partially assembled copii complex have been  tested for ability to associate with cargo protein. association of membrane-bound complex of sar1p   sec23p/sec24p with integral membrane protein indicates  that cargo proteins may laterally partition into vesicle   membrane by virtue of affinity for the Sec23p/Sec24p  protein complex (Aridor et al., 1998; Kuehn et al., 1998). early indication that the COPII coat subunits would  physically interact came from specific genetic interaction  between mutations in COPII genes. When temperature-sensitive mutation in COPII genes are combined, resulting double mutant are almost always more growth- restrictive than component single mutation, and are  usually inviable at 24 c. These 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).  specificity of type of genetic interaction suggested  synthetic lethality with known copii mutation  would be useful criterion to identify new mutation involved in the assembly of the COPII coat. We screened for mutations that were lethal with copii mutation sec13-1 and identified lst gene (lethal with sec-thirteen). As we describe elsewhere, most of  the LST genes are related to unanticipated role for  sec13 in regulated delivery of specific amino acid permeases to the cell surface (Roberg et al., 1997a,b). Accordingly, these LST genes display synthetic-lethal interactions with SEC13, but not with the other COPII genes.  On 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. Here we show that LST1 encodes homologue of the  COPII subunit, Sec24p, and that Lst1p is peripheral   membrane protein localized to the ER that can form complex with sec23p. The LST1 gene is not essential, but by  examination of phenotype of lst1Delta mutant we show  that LST1 is required for efficient export of Pma1p  from the ER to the Golgi compartment. result suggest a specialized form of vesicle coat that is responsible for recruitment of Pma1p into copii-coated vesicle. materials methods media, strains, and Plasmids The Saccharomyces cerevisiae strains used in study are listed in table   i. rich medium (ypd) and supplemented minimal medium (smm) were  prepared according to Kaiser et al. (1994). To 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 yeast culture). For experiment, SMM  was buffered to pH 6.5 using 50 mm mop and 50 mm mes. genetic manipulation were performed according to standard protocol (Kaiser et al.,  1994). dna manipulation were carried out as described in sambrook  et al. (1989). paf70 carries sec24 gene in centromere vector  pCT3 ura3; Gimeno et al., 1996). pkr34 and pKR41 carry 3.8-kb   kpni/sali fragment containing the SEC24 gene from pAF70 in 2mu vector prs426 (URA3) and pRS425 (leu2), respectively. pkr17 carries  the LST1 gene on 3.5-kb fragment in the centromere vector pRS316  (URA3). subclone of the LST1 gene from pKR17 into the 2mu vector  pRS426 gave yeast transformant at low efficiency because of toxicity of lst1 sequence when present at high copy. To study toxic   effect of lst1, pkr35 was constructed which contains the entire LST1  coding sequence expressed from pgal1 on pCD43 (URA3). pKR35 will  prevent growth under condition of induction on galactose medium,  establishing overexpression of Lst1p is toxic to yeast cells. Under conditions of partial induction of pgal1-lst1 in cell grown on raffinose,  pKR35 will complement lst1delta::leu2 for growth on acidic medium. This  shows that the LST1 open reading frame carried on pKR35 still posses  LST1 function.  Epitope-tagged LST1 was constructed as follows. First, noti site in  polylinker of pKR17 was deleted with 350-bp smai/naei fragment  pkr17delta), and then 12-bp linker carrying a NotI site (1127; new england biolabs) was inserted at eco47iii site (at codon 13 of LST1) of  pKR17Delta to make pkr17n. pkr17ha carries 100-bp noti fragment  from pgtepi (Tyers et al., 1993), which encodes tandem copy of  hemagglutinin ha1 epitope, inserted into the NotI site of pkr17n.   restriction analysis using site flanking point of insertion revealed  that 100-bp insert (ha epitope) were present in pKR17HA.  pKR17HA was transformed into cky536 to make cky535 (mata lst1Delta::  leu2 leu2-3, 112 ura3-52 [pKR17HA]). synthetic-lethal screen following plasmid and strain were constructed for use in sec13-1   synthetic-lethal screen. plasmid pkr1 carries SEC13 on 1.8-kb sali/ bamhi fragment excised from pck1313 pryer et al., 1993), inserted into  pRS316 (Sikorski and heiter, 1989). pkr4 carries the same 1.8-kb SalI/ BamHI fragment and 3.8-kb nhei/bamhi fragment containing ade3  from pdk255, both inserted into the vector pRS315 (Sikorski and Heiter,  1989). cuy563 and cky45 were crossed to produce mata ade2 ade3   leu2 ura3 sec13-1 segregant, which was transformed with pKR4 to give  cky423. mating type of CKY423 was switched by ectopic expression  of ho gene (Herskowitz and Jensen, 1991) to give cky424. culture of CKY423 and CKY424 were mutagenized by irradiation  with germicidal uv lamp at dose resulting in 10 cell survival. mutagenized cell were plated on YPD at density of 150 colony per plate.  After 5 d of growth at 24 c, colonies with solid red color and white   sector were selected for analysis. dependence of nonsectoring phenotype on sec13-1 mutation was tested by transforming candidate mutant with pkr1 or prs316. Strains that sectored after  transformation with pKR1, but not after transformation with pRS316,  were scored as sec13-1-dependent. complementation test were performed by mating mutants isolated  from CKY423 with those isolated from CKY424. zygote isolated by micromanipulation were scored for their ability to form sectored colony on  ypd plate. The genes defined by complementation group were  designated LST. lst mutant strain were backcrossed to parental   strain twice. lst sec13-1 double mutant were converted to lst single mutant by  integration of wild-type copy of SEC13 at sec13-1 locus, using the integrating plasmid p1312 (SEC13 URA3; Pryer et al., 1993). integrant  were grown on YPD and cells from white sector (indicating loss of  pKR4) were isolated. The integration of a wild-type copy of SEC13 was  confirmed by the ability of the cells from white sectors to grow at 36 c, temperature that is restrictive for the sec13-1 mutation. Owing to poor   growth of lst9 strains, we were not able to construct lst9 single mutant by  method. To test for synthetic-lethal interactions between lst mutation and mutations in sec genes, lst mutant 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 sec mutant CKY45  (sec13-1), cky50 (sec16-2), CKY78 (sec23-1), and cky450 (sec31-2). inviability of given lst sec double mutant was inferred from crosse where  lethality segregated as -gene trait tetrad giving segregation   pattern of 1:3 for lethality), outcome that was easily detectable since  crosses to wild-type typically gave >95 spore viability. The segregation  pattern of sec mutation in surviving sister spore was used as additional test to establish that inviable spore always carried the sec mutation, and therefore were not the result of random spore death. construction of lst1Delta mutants replacement of chromosomal lst1 gene with allele disrupted with  leu2 gene was constructed as follows. pKR18 carries the 5 half of  LST1 on a 2.0-kb Xho1/HindIII fragment inserted into pRS316. 2.0-kb   hindiii/bamhi fragment containing the LEU2 gene from plasmid pjj252  (Jones and prakash, 1990) and 250-bp bcli/saci fragment from 3   noncoding region of LST1 were inserted into pkr18 to construct pkr28.  nh2-terminal coding region of LST1 (except for codons 1-13) was removed by deleting 1.7-kb eco47iii/msci fragment from pKR28 to generate pkr28delta. lst1delta::leu2 construct, liberated from pKR28Delta by digestion with xhoi, was transformed into wild-type diploid strain   cky348 mata/alpha leu2-3,112/leu2-3,112 ura3-52/ura3-52). On sporulation  and dissection, this diploid gave viable spore clone, and haploid segregant carrying lst1Delta::LEU2 were confirmed by southern blotting. segregant was further backcrossed to s288c genetic background  to give strains CKY536 and CKY542. proton efflux from intact yeast cells pma1p activity was assayed by proton efflux from intact cell into external medium. Cells were grown to exponential phase in YPD at 37 c,  washed, and then stored in deionized water at 4 c overnight. cell number  was measured by light scattering, and total of 25 a600 unit (~5 x 108   cell) was suspended in 5 ml of 100 mm kcl, 10 mM glycine, pH 4.0. The  pH of cell suspension was measured using a combination electrode at  25 c with constant stirring. Once the pH had stabilized (~10 min), glucose was added to final concentration of 40 mM and ensuing drop in  pH was recorded at 30-s interval over 15 min. In comparison of wild-type  (cky443) and lst1Delta (CKY536) strains, suspension had identical cell   concentration as measured by light scattering, and showed response to calibration pulse with HCl. immunofluorescence microscopy intracellular location of Pma1p in wild-type (CKY443) and lst1Delta  (CKY536) cells was examined by indirect immunofluorescence microscopy using technique described previously (Pringle et al., 1991; Espenshade et al., 1995). Strains were grown exponentially in smm medium, pH  7.2, at 30 c. cells were fixed in 3.7 formaldehyde and then converted to  spheroplast by digestion with lyticase. primary secondary antibody incubation were for 1 h at 25 c. affinity-purified anti-pma1p antibody was prepared as follows. crude preparation of yeast membrane  was resolved by preparative sds-page, and after transfer of proteins to  nitrocellulose membrane by electrophoresis, strip of membrane that  contained Pma1p was excised. rabbit antiserum to Pma1p was applied to  nitrocellulose strip, and after the strip was washed with 20 mm tris   ph 7.5 150 mm nacl, 0.5 tween 20, bound antibody was eluted  with 100 mM glycine, ph 2.8, 500 mM NaCl, 0.5% Tween 20. affinity-purified pma1p was used at 1:100 dilution and fitc-conjugated anti-rabbit   igg was used at 1:200 dilution. mounting medium was supplemented with  4,6-diamidino-2-phenylindole (dapi). micrographs were taken with nikon eclipse te300 microscope with hamamatsu orca c4742-95 ccd   camera. For localization of lst1p-ha, CKY535 was grown on SMM to exponential phase and prepared as described above. For visualization of  Lst1p-HA, 12ca5 antibody (berkeley antibody co., inc.) was used at  1:5,000 dilution and fitc-conjugated goat anti-mouse igg was used at  1:50 dilution. rabbit anti-kar2p polyclonal serum (gift of M. Rose,  princeton university, Princeton, nj) was used at 1:1,000 dilution and  rhodamine-conjugated goat anti-rabbit igg was used at a 1:200 dilution.  sample were viewed and imaged using nikon optiphot 2 microscope  and photometric imagepoint ccd camera. image were recorded using  ip-lab software (molecular dynamics, Inc.). cell fractionation cell organelle were fractionated on equilibrium density gradient as previously described (Roberg et al., 1997a). Cultures were grown exponentially at 24 C and then shifted to 37 C for 3 h. 1.6 x 109 cell 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 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 bead. additional 1 ml of ste10  was added, and lysate was cleared of unbroken cell and large cell debris by centrifugation at 300 g for 2 min. cleared extract (300 mul) was  layered on top of 5-ml 20-60 linear sucrose gradient in te (10 mm   tris-hcl, pH 7.6, 10 mM EDTA) prepared for sw50.1 rotor (beckman instruments, Inc.). Samples were centrifuged 100,000 g for 18 h at 4 C  and fraction of 300 mul were collected from the top of gradient. Protein  was precipitated from each fraction by addition of 100 mul of 0.15  deoxycholate and 100 mul of 72 trichloroacetic acid. protein pellet 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). Pma1p, gas1p, and sec61p were  resolved by sds-page and were detected by immunoblotting. relative of each protein in cell fraction was determined by densitometry using ultroscan 2202 lkb instruments, Inc.). golgi gdpase   activity was assayed in gradient fraction before protein precipitation using standard method (Abeijon et al., 1989). subcellular distribution of Lst1p-HA was examined using techniques described previously (Espenshade et al., 1995). CKY535 carrying  pkr17ha, which expresses Lst1p-HA, was grown to exponential phase  in SMM without uracil. 2 x 109 cell 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 protease   inhibitor cocktail. 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 soluble particulate fraction. release of Lst1p-HA from 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. After 1 h of incubation at 4 C, samples were centrifuged at 50,000 g for 30 min to separate soluble and particulate fractions. Fractions from both experiments were solubilized in sample buffer and analyzed by immunoblotting. immunoblotting sample of 10-30 mul in ESB were resolved by SDS-PAGE and immunoblotting was conducted according to standard protocols (Harlow and  Lane, 1988). For transfer of Lst1p to nitrocellulose membranes, 0.1 sds  was included in transfer buffer. following antibody were used:  mouse monoclonal 12ca5 anti-ha at 1:1,000 dilution; rabbit anti-pma1p   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 hrp-coupled sheep anti-rabbit ig (nycomed amersham corp.) at 1:10,000 dilution. blot were developed using chemiluminescence detection system (Nycomed Amersham Corp.). Pulse-Chase Kinetics of invertase maturation The strains used for radiolabeling all carried plasmid pnv31, which  carries suc2 gene under constitutive tpi1 promoter (a gift of M.  Lewis, medical research council laboratories of molecular biology,  Cambridge, uk). Wild-type (cky540) and lst1Delta (CKY542) strains were  grown in SMM without methionine (buffered with 50 mM MES and 50 mm   mop to pH 6.5) at 24 C to exponential phase, and then shifted to 37 C  for 3 h before labeling. 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. radiolabeling and immunoprecipitation of invertase was  performed as previously described (Gimeno et al., 1995; elrod-erickson  and Kaiser, 1996). -hybrid interactions yeast-hybrid assay was used to test potential protein-protein interaction as previously described (Gyuris et al., 1993; bartel and Fields,  1995). interaction were tested between either Lst1p or Sec24p fused to  lexa dna-binding domain and Sec23p fused to acidic transcriptional activation domain. The following plasmids were used: ppe81 carries SEC23 fused to acidic activation domain of pjg4-5 (Espenshade  et al., 1995); prh286 carries sec24 (codons 34-926) fused to lexa   dna-binding domain in peg202 (Gimeno et al., 1996); pkr37 carries  LST1 fused to the lexA DNA-binding domain in pgilda (derivative of  pEG202 with pGAL1; provided by D. Shaywitz). Combinations of control fusion protein plasmid, along with reporter plasmid psh18-34, were transformed into the strain egy40 (golemis and brent, 1992). Strains were grown exponentially in SMM with 2%  raffinose as carbon source. Galactose was added to concentration of  2%, and incubation was continued for 10 h to induce fusion protein expressed from pGAL1. assay for beta-galactosidase activity were performed  on cells lysed by disruption with glass beads (rose and botstein, 1983).  activity was normalized to total protein determined by the Bradford assay bio-rad laboratories). Binding of Lst1p to Sec23p gene fusion expressing Lst1p fused to glutathione s-transferase  (gst) was constructed by inserting 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. ppe123  is sec23 gene expressed from pGAL1 in prs315 (Gimeno et al., 1996).  binding interaction were tested from extract of cky473 transformed with  pRH254 (GST-Lst1p-HA) and either pCD43 (vector) or pPE123 (Sec23p). Cells 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. 5 x 108 cell 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. The extract was diluted to 1 ml with IP buffer, and membranes were collected by centrifugation at 500 g for 20 min. pellet was  extracted with 1 ml of IP buffer and 600 mm nacl for 10 min at 0 c to release membrane-bound protein complex. After clarification by centrifugation at 90,000 g for 10 min, the extract was diluted threefold with ip   buffer, and 1-ml aliquot was removed and incubated at room temperature for 1 h with glutathione Sepharose 4B beads (Pharmacia Biotech,  Inc.). bead 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. Proteins were released  from glutathione sepharose 4b bead by solubilization in ESB. Samples  of total lysate were prepared by adding 2x ESB to equal of diluted extract from salt washed membranes. Samples were analyzed  by immunoblot probed with anti-sec23p antibody. For analysis of membrane association of gst-lst1p-ha   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. 2 h after induction, 2 x   107 cell 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. Cells were lysed by vigorous agitation with glass beads  and an additional 500 mul of lysis buffer was added. The lysate was cleared of  unlysed cell and large cell debris by centrifugation at 300 g for 3 min 50 mul  of supernatant was reserved for total extract sample and remainder  was centrifuged to pellet ER membranes at 10,000 g for 30 min at 4 C in microcentrifuge. equal number of cell equivalent of total extract, membrane-pellet, and supernatant fraction was solubilized in ESB and analyzed  by immunoblotting. cytosolic protein gdh2p was found only in soluble fraction, demonstrating cell lysis was complete (datum not shown). Results Mutations Synthetically Lethal with sec13-1 To find new gene required for budding of copii vesicle, we screened for mutations that displayed synthetic   lethality with the COPII mutation sec13-1 using plasmid   sectoring assay (Roberg et al., 1997b). Strain CKY423 has  chromosomal mutation ade2 ade3 sec13-1 and harbors  plasmid pkr4, which carries wild-type copies of  SEC13 and ADE3. This strain accumulates red pigment  because of ade2 mutation, but spontaneous loss of  pKR4 during the growth of colony gives white sectors of  ade2 ade3 segregants. In this strain, a mutation that is lethal with sec13-1 will produce nonsectoring colony. mutagenesis of CKY423 and isogenic strain of opposite   mating type, CKY424, yielded 139 nonsectoring mutant  (Fig. 1). These strains were then tested for restored ability  to sector after transformation with pKR1, which carries  wild-type sec13, but lacks ade3 gene. By test, 57  of the mutants had synthetic-lethal mutation that could  be rescued by wild-type SEC13. In backcrosse, 52 mutant gave segregation pattern indicating that trait  was due to single nuclear mutation (Fig. 1).  mating between mutants identified 11 complementation group using colony sectoring of the diploid as criterion for allelic complementation. These complementation groups were designated LST (table ii). One of the  complementation groups was shown to comprise recessive   lethal mutation in sec13 gene itself (Roberg et al.,  1997b). Tests for rescue of the nonsectoring phenotype by  plasmid carrying known sec gene showed that LST10  was SEC16 (Roberg et al., 1997b).  synthetic interactions of lst Mutations To perform genetic test 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). representative lst   single mutant were then crossed to sec16, sec23, and sec31   mutant. For 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). We have subsequently shown that lst   gene relate to a function of SEC13 in sorting of amino   acid permease in late secretory pathway, and analysis  of these genes is described elsewhere (Roberg et al.,  1997a,b). Mutations in LST1 were inviable when combined with sec16, sec23, and sec31 mutation, and mutations in LST6 were inviable with sec16 and sec31 table   iii). Importantly, mutations in LST1 and LST6 did not  show synthetic lethality in parallel crosse to mutations in  sec17 or sec18, genes required for fusion of COPII vesicles. Given that synthetic-lethal interactions usually occur  between mutations in genes involved in step of  the secretory pathway, the tests for genetic interactions indicated that LST1, and probably lst6, participate in  vesicle budding from the ER.  Lst1p Is Homologous to Sec24p The 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 presence of lst1-1 mutation.  CKY426 was transformed with yeast genomic library. 34   colony that regained the ability to form white sectors  were identified among 97,000 ura+ transformant. We expected screen to yield plasmids carrying either SEC13  or LST1. About half of complementing plasmid were  shown to carry SEC13 by restriction site mapping and by  the ability to complement temperature sensitivity of  sec13-1. restriction map of remaining rescuing   plasmid show that they represent unrelated chromosomal region. clone p21-31 and p77-2 were selected  as representative of region. genomic sequence  from p77-2 (a clone in plambdayes vector; elledge et al.,  1991) was inserted as xhoi fragment into integrating vector prs306 to produce pkr20. For chromosomal   integration, pKR20 was linearized by digestion with HpaI  and transformed into cuy564 matalpha ade2 ade3 leu2   ura3). resulting strain was crossed to the lst1-1 mutant  CKY426 (mata lst1-1 sec13-1 ade2 ade3 leu2 ura3  [pKR4]). After sporulation and dissection, integrated   pkr20 was found to be completely linked to the LST1 locus: sectoring segregated 2:2 and all sectored colonies  were Ura+, whereas nonsectored colony were ura-.  Thus, p77-2 carries the LST1 gene. In parallel, the genomic sequence from p21-31 (a clone in pct3 vector;  Thompson et al., 1993) was inserted as ecori/hindiii   fragment into prs306 to produce pkr7. pKR7 was integrated at chromosomal locus after linearization with  MscI and was then checked for linkage to lst1-1. tetrad   analysis showed that pKR7 was not linked to LST1 and we  concluded that pKR7 carries unlinked suppressor gene. 3.5-kb insert of p77-2 was inserted into xhoi   site of centromeric vector pRS316 to construct pKR17.  base sequence of insert was determined and  found to contain single open reading frame encoding a  protein of 929 amino acid. This sequence corresponds to  open reading frame yhr098c located on chromosome   viii (saccharomyces genome database, Cherry et al.,  1997). predicted amino acid sequence of LST1 shows  significant similarity to SEC24 (yil109c). The two proteins share 23 sequence identity that extends over most  of length (Fig. 2), suggesting that Lst1p may have a  function similar to that of Sec24p as subunit of copii vesicle coat.  phenotypes of lst1Delta copy of the LST1 gene in wild-type diploid strain  cky348 was disrupted to generate lst1delta::leu2/lst1   heterozygote. Sporulation and dissection of this diploid  gave >95% spore viability on ypd medium and leu2 marker segregated 2:2, showing that LST1 is not essential for growth. lst1delta::leu2 mutant spore clone was  crossed to sec mutants to test for synthetic lethality. In  these crosses, both the temperature sensitivity of sec   mutation and lst1delta allele marked by LEU2 could be  followed independently. In crosses of lst1Delta to sec12, sec13,  sec16, sec23, sec24, or sec31 mutant, inviability segregated  as a two-gene trait (segregation patterns for dead viable   spore clone were 2:2, 1:3, and 0:4). Tests of genotype  of surviving sister spore clones showed that the inviable spores in these crosses were always lst1Delta sec double   mutant. Crosses between lst1Delta and sec17 or sec18 produced viable double mutant. These findings confirmed  and extended earlier test for synthetic lethality with  lst1-1, and demonstrated that lst1Delta was synthetically lethal  with known gene required for COPII vesicle formation, but not with genes required for vesicle fusion. We evaluated the growth of lst1Delta::LEU2 mutants under  variety of conditions. On YPD, lst1delta::leu2 strain  grew, as well as an isogenic wild-type strain at temperatures ranging from 14 to 37 c. However, on SMM the  lst1Delta::LEU2 strain grew poorly at temperatures above  30 c. Since YPD (pH 6.5) and SMM (pH 3.8) differed  markedly in pH, we suspected that lst1Delta mutants may be  particularly sensitive to acidic environment, and we  tested effect of pH on the growth of lst1Delta mutants. Although lst1Delta mutants grew as well as wild-type on YPD at  all temperatures, when YPD was brought to pH 3.8, lst1delta   mutant grew much more slowly than wild-type at 37 C  (Fig. 3 A). Conversely, on SMM buffered to pH 6.5, lst1Delta  and wild-type grew even at 37 C (data not shown). These  results demonstrated that at high temperature, growth of  the lst1Delta mutant was sensitive to acidic condition.  Having identified conditions where LST1 was needed  for growth, we investigated whether overexpression of  SEC24 could supply the function lost in lst1Delta. restoration of function was indicated by the ability of an lst1Delta  mutant to grow on acidic medium when provided with extra copy of SEC24 on centromeric 2mu plasmid  (Fig. 3 b). These findings imply functional overlap  between LST1 and SEC24. In parallel test for suppression, we found that the genes SEC12, SEC13, sec31, or  SEC23, when expressed from 2mu plasmid, could not restore the ability of an lst1Delta mutant to grow on acidic medium. We found that lst1delta mutation caused selective   defect in trafficking of Pma1p from the ER, and we  also examined the ability of overexpressed sec24 to suppress this phenotype caused by the lst1Delta mutation. By immunofluorescence microscopy, proper localization of  Pma1p to the cell surface was restored in lst1delta strain  that also carried SEC24 on a 2mu plasmid (see Fig. 5).  In attempt to test the effect of overexpression of  LST1, we found that LST1 on a 2mu plasmid severely impaired growth of wild-type yeast cell. To examine the response of cells to different dose of Lst1p, we designed way to express different level of Lst1p according to the  amount of galactose in growth medium. wild-type   strain (CKY473) carrying a plasmid that expressed LST1  from pGAL1 (pKR35) was spread on smm plate with  2% raffinose, a carbon source that allows yeast growth  without repression of gal1 promoter. When these  cells are exposed to a gradient of galactose concentration,  from 3 mg of galactose in filter disk on top of lawn,  growth was inhibited in halo 1.5 cm beyond edge of  filter (Fig. 3 C). A strain that did not contain pKR35  grew uniformly up to the edge of the filter, showing that  the galactose itself was not inhibitory. Given similarity  of Lst1p to Sec24p, we asked whether the overexpression  of SEC24 could compensate for overexpression of LST1.  Cells carrying both the pGAL1-LST1 plasmid (pKR35)  and the SEC24 gene on a 2mu plasmid (pkr41) were tested  in identical halo assay, and were found to be resistant to  the effect of galactose (Fig3 c). Suppression by SEC24  appeared to be specific, since parallel tests of 2mu plasmids  carrying SEC12, SEC13, SEC31, or SEC23 failed to show  suppression. It is worth noting that SEC23 expressed from  2mu plasmid significantly slows the growth of yeast  strains. Any suppression afforded by overexpression of  SEC23 might be counteracted by inherent toxicity of  SEC23. simple conclusion that can be drawn from overexpression study is that too great of stoichiometric   excess of Lst1p over Sec24p is lethal. observation can  be explained if Lst1p and Sec24p compete with one another in the assembly of vesicle coat complex and that  excess lst1p causes sequestration of vesicle component  into complexes that fail to satisfy essential function  of COPII. lst1Delta Diminishes the Activity of the Plasma Membrane Proton-ATPase sensitivity of lst1Delta mutants to low pH suggested involvement of Pma1p, which has been shown to be limiting cell component for growth on acidic medium (McCusker et al., 1987; Portillo and Serrano, 1989). The dependence of Pma1p activity on LST1 was supported by the  observation that lst1Delta mutants exhibited unusual morphology characteristic of pma1 mutant. When lst1Delta mutants were grown in low pH (SMM or YPD brought to pH  3.8) at 37 C, ~10% of the cells formed multibudded rosette; in case, 15 daughter radiated  from single large mother cell (Fig. 4 A). unseparated   daughter cell contained nucleus that could be stained with  DAPI and daughter cell could be separated from  mother by micromanipulation, indicating they had  completed cytokinesis. Cells depleted of Pma1p produce  similar multibudded cell with attached daughter that had  completed cytokinesis. In this case, multibudded rosettes  are thought to form because 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 bud  themselves (Cid et al., 1987). morphology of lst1delta   cell 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.  As direct test of the effect of lst1Delta on the activity  of Pma1p, we measured capacity of mutant cell to  pump protons into the external medium. wild-type   lst1delta strain were cultured in YPD at 37 C, conditions under which both strains grow equally well. After starvation  by prolonged incubation in water, the cells were placed in  weakly buffered medium proton efflux on addition  of glucose was measured as drop in extracellular ph. For  wild-type lst1delta strain, addition of glucose produced sharp decline in pH (after 30-s lag), which began  to level off after ~5 min (Fig. 4 b). Although the responses  of wild-type lst1delta cell were qualitatively similar proton efflux from lst1delta cell was compromised: in 5 min after addition of glucose rate of change in pH  produced by the lst1Delta mutant was 65% of that of wild-type.  These findings indicate that the lst1Delta mutant grown at 37 C  has about half of the Pma1p activity as wild-type cell. LST1 Is Required for Efficient Transport of Pma1p Out of the ER To determine whether reduced pma1p activity in lst1Delta  mutants was due to defect in the transport of Pma1p to  the cell surface, we compared the localization of Pma1p in  wild-type lst1delta mutant cell by immunofluorescence   microscopy. Cells were grown at 30 C in YPD medium to  avoid possible secondary effect due to the pH sensitivity  of lst1Delta mutants. In lst1Delta cells, Pma1p was located primarily at nuclear periphery and at cellular rim, indicating that large proportion of Pma1p remains in the ER  (Fig. 5). pattern of localization differed markedly  from 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). We also examined the subcellular distribution of Pma1p  in lst1Delta cells by cell fractionation. Lysates from cells grown  at 37 C for 3 h were fractionated on sucrose density gradient under conditions where er plasma membrane are well separated on basis of buoyant density. Pma1p from wild-type cells was located in dense   fraction of the gradient in peak that was coincident with  that of Gas1p, gpi-linked plasma membrane protein  (Nuoffer et al., 1991). In contrast, <35% of total   pma1p from lst1Delta cells coincided with the plasma membrane marked by gas1p protein and majority of  Pma1p was located in fractions containing the ER (Fig. 6).  Interestingly, the ER from lst1Delta mutants (marked by  Sec61p) reproducibly resolved into two peaks of different   density, suggesting accumulation of pma1p segregates   er membrane into subdomain of relatively high low   density. Given that most of the Pma1p was located in er peak of higher density, it is possible that the density of  the ER had been increased because of the accumulation of  Pma1p. similar increase in density of portion of the  ER is caused when folding mutants of pma1 are retained  within the ER (Harris et al., 1994).  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.  absence of general protein secretion defect in lst1Delta  mutants was implied by normal growth of lst1Delta mutants at 37 C in medium of pH 6.5 (doubling time of  lst1delta wild-type was 1.75 h in YPD), indicating normal rate of expansion of the plasma membrane. As specific test for the rate of ER to golgi transport, pulse- chase experiment were performed to follow the rate of  maturation of invertase from core glycosylated er   form to golgi and secreted form. 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). Similarly, no defect in the maturation of carboxypeptidase y from er form to the  Golgi and vacuolar form of enzyme could be detected  (data not shown).  We also considered possibility that transport of  Pma1p may be particularly sensitive to subtle defect in  vesicle formation. We addressed this possibility by examining the localization of Pma1p in sec24-1 sec31-2 mutant cell at semipermissive temperature of 28 c. Although 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 perinuclear   region of either mutant by immunofluorescence microscopy (data not shown). Thus, partial defect in copii   function did not lead to extensive accumulation of  Pma1p in the ER that was observed for lst1Delta mutants.  Taken together, comparisons between the lst1Delta mutation  and copii gene mutation indicate that the lst1Delta mutation is unusual in its ability to inhibit pma1p exit from the  ER without interfering with the transport of cargo   protein. Localization of Lst1p To examine intracellular distribution of Lst1p, epitope-tagged derivative was constructed by inserting six  copies of 10-amino acid ha near nh2 terminus of  Lst1p. ha-tagged lst1 was functional, as demonstrated by its ability to complement lst1-1 in sectoring assay, and to restore the ability of a lst1Delta mutant to grow on  acidic medium at 37 C (not shown). In cells expressing  Lst1p-HA that were fixed for immunofluorescence microscopy, staining was found primarily at the nuclear periphery (Fig. 8). signal was seen in cells expressing untagged lst1p, verifying that origin of staining   pattern was due to Lst1p-HA. Although Lst1p-HA staining largely coincided with er marker kar2p, there  were subtle difference in their patterns of localization:  kar2p appeared uniformly, distributed around nuclear   periphery, whereas lst1p-ha staining had punctate appearance indicating that Lst1p might be concentrated in particular region of the ER. In addition, weak   punctate staining was observed throughout cell body,  some of which may correspond to ER membranes near cell periphery.  The intracellular distribution of Lst1p was also examined by subcellular fractionation. Cells expressing Lst1p-HA were converted to spheroplasts and then gently lysed.  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). All of soluble marker protein   gdh2p (Miller and Magasanik, 1990) was found in the  150,000 g supernatant fraction, indicating complete cell lysis (data not shown). The association of lst1p protein  with sedimenting fraction was analyzed by chemical   treatment of cell lysates before centrifugation at 50,000 g.   incubation 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). partial dissociation of  Lst1p-HA from the sedimenting fraction by agent  suggested that Lst1p is peripheral membrane protein  that adheres tightly to the membrane.  lst1p binds sec23p sec24p was first identified as a protein that formed 400-kd   complex with Sec23p (Hicke et al., 1992). Because of the  similarity of Lst1p to Sec24p, we investigated whether  Lst1p could also bind to Sec23p. To assay potential interaction 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). Interaction  between the two fusion proteins was tested by assaying for  activation of lacz reporter gene. Induction of beta-galactosidase was observed when lst1 sec23 fusion  were coexpressed, but not when expressed alone (Table  IV). 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).  To confirm the interaction between Lst1p and Sec23p,  association of these proteins was examined in yeast cell extract. coding sequence of lst-1-ha (codons 14-927)  was fused to GST and expressed in yeast from pgal1   promoter. SEC23 was also expressed from pGAL1. Since  both proteins are largely associated with intracellular   membrane (Fig. 10 b), membranes prepared from cells  overexpressing sec23p gst-lst1p-ha were  first extracted with 600 mM NaCl to release protein complex from the membrane, salt extract were clarified  by centrifugation at 90,000 g, and diluted to give final   concentration of 200 mM NaCl. GST-Lst1p-HA was  isolated from the extracts by affinity to glutathione   sepharose bead. Sec23p was found in association with  gst-lst1p-Ha, but not in control extract prepared from  cells expressing Sec23p and GST alone (Fig. 10 A). Together, these experiments show that Lst1p, like Sec24p,  can form a complex with Sec23p.  Sec23p and Sec24p have been shown to assemble onto  the ER membrane as a complex (Matsuoka et al., 1998).  While working out conditions to optimize recovery of  Sec23p bound to GST-Lst1p-HA, we discovered that assembly of lst1p/sec23p complex appears to enhance  the association of both proteins with the ER membrane.  When 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). This pellet contains most  of the ER, as marked by er membrane protein  Sec61p (data not shown). When material that pelleted at  10,000 g was suspended in 60 sucrose and applied to the  bottom of a sucrose density gradient, >90% of gst- lst1p-ha and Sec23p cofractionated with 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). In 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 strain overexpressing sec23p alone. Similarly, < 20 of the GST-Lst1p-HA pelleted at 10,000 g in  lysates from a strain expressing GST-Lst1p-HA alone  (Fig. 10 B). Thus, when either Sec23p or GST-Lst1p-HA  was overexpressed alone, most of overexpressed protein was soluble, but when the proteins were expressed together, most of the proteins were associated with the ER  membranes. These data support the observation that Lst1p  can form a complex with Sec23p, and that lst1p/ sec23p complex has affinity for ER membranes. discussion By screening for mutants that exhibited synthetic-lethal   genetic interaction with the COPII mutation sec13-1, we  identified the LST1 gene. subsequent genetic test 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). This pattern of genetic interactions  indicated that LST1 participates in process of vesicle  budding from the ER, expectation that was born out by  the examination of the LST1 gene and product. following observation indicate a role for Lst1p as part of copii-like vesicle coat: (I) LST1 encodes 90-kd protein  that is homologous to copii-coat subunit sec24p. The  two proteins share 23 amino acid identity over entire length. ii lst1p is a peripheral ER membrane protein as shown by immunofluorescence microscopy and cell   fractionation. iii lst1p, like Sec24p, can bind to Sec23p  as shown by tests for -hybrid interaction and affinity purification of a complex of GST-Lst1p and Sec23p.  iv assembly of sec23p-lst1p complex appears to  enhance the membrane association of lst1p   sec23p: when both proteins are overexpressed together,  most associate with membranes, whereas either protein  overexpressed alone is mostly cytosolic. (V) Although  strains with chromosomal deletion of LST1 are viable and  appear normal for secretion of marker protein, these mutants show pronounced accumulation of Pma1p in the  ER, indicating selective defect in ER to golgi traffic.  Based on these findings, we propose that Lst1p takes the  place of Sec24p in specialized copii coat complex that  is used for the recruitment of Pma1p into vesicles. Strains carrying lst1Delta have phenotypic hallmark of  deficiency in Pma1p activity, including sensitivity to  growth in an acidic environment, formation of multibudded cell, and decreased rate of proton efflux from  intact cells. All three traits are expressed only at temperatures of 30 C and above, indicating that LST1 is only required for Pma1p activity at high temperature. Localization 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. export of Pma1p from the ER cannot be completely dependent on Lst1p, since Pma1p transport appears normal  in lst1Delta mutants at 24 C. Even at 37 C, block in pma1p   transport may not be complete ~35 of total   pma1p fractionate with the plasma membrane, although  some of the Pma1p detected in the plasma membrane in  this experiment was probably synthesized before shift  to restrictive temperature. Therefore, it seems likely that  Lst1p and Sec24p share burden of transporting Pma1p  from the ER. At 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. transport defect caused by deletion of LST1 appears to be specific for Pma1p. Under conditions where a  defect in Pma1p transport was observed in lst1Delta mutants,  transport of Gas1p, carboxypeptidase Y, and invertase  was unaffected. Using growth as general assay for  trafficking defect, we found that lst1Delta mutants grew at identical rate to wild-type at 37 C when we compensated  for the defect in Pma1p transport by using media at pH  6.5. This indicates that rate of expansion of plasma   membrane, including the transport of essential plasma   membrane protein, is not significantly affected by the absence of LST1. We also considered the possibility that there may be difference among cargo molecules in their response to general defect in protein transport machinery. Of 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 standard marker protein might have significant   effect on the rate of transport of Pma1p. If this were the  case, partial defects in copii component should  also interfere with Pma1p transport. Therefore, we examined sec24 and sec31 mutants, but could find evidence  for a defect in Pma1p transport, even at semipermissive   temperature where the rate of growth was inhibited. Although Pma1p was essential protein for which we  could detect a transport defect in lst1 mutant, a defect in  the transport of nonessential protein could have been  overlooked by our analysis. factor required for the transport of specific membrane   protein have been documented in number of other  cases. shr3 gene encodes 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). A set of er protein, Vma12p, vma21p, and  vma22p, are required for transport from the ER of integral membrane subunit of the vacuolar atpase (hill  and Stevens, 1994, 1995; jackson and stevens, 1997). Similarly, mutational study have shown that small er   membrane protein erv14p is specifically required for  transport of plasma membrane protein Axl2p out of  the ER (powers and Barlowe, 1998). Finally, 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). In all of these  cases, question remains whether Shr3p, the Vma proteins, Erv14p, or Ast1p act directly in vesicular transport of  respective cargo molecule, or whether they are primarily involved in protein folding and influence protein  sorting indirectly through quality control mechanism. Because Lst1p appears to be component of a vesicle coat,  Lst1p seems more likely to have direct role in the sorting  of Pma1p rather than in folding. Expression of a variety of dominant pma1 mutation  can cause accumulation of mutant wild-type   pma1p in proliferated er harris et al., 1994; Portillo,  1997). Similarly, the transport of wild-type pma1p from  the ER is blocked when pma2 (isoform of PMA1)  or plant plasma membrane proton-atpase are overexpressed in yeast (villalba et al., 1992; Supply et al., 1994;  de kerchove dExaerde et al., 1995). proposal was  that special factor may be required for the transport of  Pma1p from the ER in manner analogous to requirement for shr3p in the transport of amino acid permeases  (supply et al., 1994). specific role of Lst1p in the  transport of Pma1p suggests that it may be the factor depleted by the expression of dominant form of Pma1p. In  future, it may be possible to test idea by evaluating  the ability of lst1p overexpression to reverse the effects of  dominant PMA1 mutations. mechanism by which Lst1p acts in the transport of  Pma1p may be inferred from recent study examining the  recruitment of cargo molecules into COPII vesicles. Using  er-derived microsome and purified copii component,  Kuehn et al. (1998) have shown that the Sec23p/Sec24p  complex, along with Sar1p, associate with amino acid permease and other integral membrane protein that are destined for the plasma membrane. In parallel experiment  using mammalian microsomes, mammalian sec23p/sec24p  and Sar1p were found to bind to microsomal membrane  and form a complex that contains the cargo protein VSV-G  (Aridor et al., 1998). conclusion from experimental system is that sec23p/sec24p complex contains specific binding site for capture of membrane   cargo protein within plane of the ER membrane.  Based on the data presented here, Lst1p appears to be an  isoform of Sec24p that is adapted for selection of Pma1p.  This provides the first evidence that Sec24 family members carry information specifying the type of cargo molecules that are accepted by er-derived vesicle. We have looked for association of Lst1p with ER-derived vesicles, but under the conditions of vitro   budding reaction, large quantity of Lst1p-HA is released  from the membrane in soluble form. soluble lst1p-ha  gives high background in vesicle fraction preventing us  from reliably determining whether there is specific association of Lst1p with vesicles. In future experiment, it  may be possible to isolate vesicles coated with Lst1p by  performing vitro budding reaction using purified cytosolic component, including purified complex of Lst1p  and Sec23p. It may also be possible to determine whether  vesicles that are formed using sec23p/lst1p complex   efficiently incorporate pma1p than vesicles formed  using the Sec23p/Sec24p complex. Finally, it will be of interest to determine if there is direct binding of Lst1p to  Pma1p. identification of sec24p homologue that also acts  in transport from the ER raises the possibility that coat of ER-derived vesicles may be heterogeneous. It is  possible that sec23p/lst1p complex act to form class of  vesicle that is distinct from those formed by sec23p/ sec24p complex. Alternatively, it is possible that the two  complexes assemble together forming vesicles with coats  of mixed composition. The identification of additional homologue of Sec23p and Sec24p suggest existence of  coats with greater combinatorial complexity. We  have identified sec24p family member, which we  call iss1p, as a protein that binds to Sec16p. Iss1p  (YNL049c) also binds Sec23p and appears to be associated  with the ER membrane (gimeno, 1996). In addition, saccharomyces genome contains uncharacterized open   reading frame (yhr035w) that is 21% identical to Sec23p  (Saccharomyces Genome Database, Cherry et al., 1997). If  each of the Sec23p and Sec24p homologues carry different   determinant for cargo selection, and if mixed coat can  form, possible combination of sec23p sec24p homologue should allow the formation of wide variety of  copii-like vesicle with different capacity to carry different cargo molecule. figure and Tables S. cerevisiae Strains     Strain  Genotype  Source or reference    cuy563  mataade2-101 ade3-24 leu2-3,112 ura3-52   t. huffaker (cornell university)  CUY564  MATalpha ade2-101 ade3-2 leu2-3,112 ura3-52  T. Huffaker (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 ura3-52   All strains are from this study unless otherwise indicated.      colony-sectoring screen for mutations that are lethal  with sec13-1. CKY423 (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 red colony. Mutagenized cells that have acquired lst   mutation cannot grow without the pKR4 plasmid form nonsectoring solid red colony. Of 132 nonsectoring colony, sectoring in 57 was restored by transformation with second   sec13-bearing plasmid (pKR1). Mutations Lethal with sec13-1      gene   number of alleles   LST1  11   lst2   6  LST3   4  LST4   5  LST5   5  LST6   1  LST7   1  LST8   1  LST9   1  LST10 (SEC16)   2 Growth of lst sec Double Mutants at 24 C       sec13-1  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   nd growth is represented in decreasing order by: + > +/- > +/-- > -. nd, not determined.              Comparison of LST1  and sec24 sequence. identity  are indicated by solid line and  similarities are indicated by dotted line. overall amino acid   identity is 23%. functional relationship between LST1 and SEC24.  (A) Sensitivity of lst1Delta mutants to acidic medium. Equal 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). Plates were photographed after incubation at 37 C for 2 d. (b) lst1delta::leu2   strain (cky552) was transformed with: vector only, pRS316;  LST1 on centromeric plasmid, pKR17; SEC24 on a centromeric  plasmid, pAF70; or SEC24 on a 2mu plasmid, pKR34; and  streaked onto YPD medium, pH 3.8. Colonies were photographed after growth at 37 C for 2 d. (c) 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). transformant  were plated at a density of 800 cell/cm2 on SMM plates containing 2% raffinose and then 3 mg galactose solution was placed on  sterile 1-cm filter on top of the lawn. The plates were photographed after growth at 30 C for 2 d. Pma1p accumulates the ER  in lst1Delta cells and this accumulation is  suppressed by overexpression of SEC24.  Cells 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. field of cells,  stained with DAPI to label nuclear   dna, are also shown. panel, montage of lst1Delta cells (CKY536 carrying vector prs316); middle panel,  genotypically wild-type cell (CKY536  carrying the LST1 plasmid pKR17); bottom panels, lst1Delta cells suppressed by  SEC24 (CKY536 carrying the 2mu SEC24  plasmid pkr34). bar, 5 mum. pma1p defect  caused by lst1Delta. (A) lst1Delta  cells (cky534) were photographed using differential interference contrast microscopy after growth at 37 C on  YPD, pH 3.8. A montage of  multibudded cells is shown.  Cells of type comprise  ~10% of lst1delta culture, but  are never seen in wild-type  grown under the same conditions. Bar, 10 mum. b reduced capacity for proton   pumping by lst1Delta cells. Wild-type (CKY443) and lst1Delta  (CKY536) were grown to  exponential phase in YPD  medium, pH 6.8, at 37 c.   cells were incubated in water overnight and then suspended in 10 mM glycine  buffer at pH 4.0. Proton efflux from the cells after addition of glucose was recorded as decrease in the pH of the external medium. Based on 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. Cell fraction to localize Pma1p in lst1Delta cells. Wild-type  (CKY443) and lst1Delta (CKY536) cells were grown in YPD at 24 C  and then were shifted to 37 C for 3 h. cell lysate were fractionated on density gradient of 20-60 sucrose. relative level of  Pma1p, Gas1p (plasma membrane marker), and sec61p er   marker) in each fraction were quantitated by immunoblotting  and densitometry. gdpase golgi compartment marker) was determined by enzymatic assay. Transport of invertase is not affected by lst1Delta. Wild-type (CKY540), lst1Delta (cky542), and sec12-4 (CKY541) strains  expressing invertase from constitutive ptpi1-suc2 fusion,  were grown to exponential phase at 24 C in SMM medium, pH  6.5, without methionine. Wild-type and lst1Delta strains were shifted  to 37 C, grown for 3 h, and sec12-4 cky541 strain was  shifted to 37 C 5 min before labeling. Cells were pulse-labeled with  [35S]methionine and cysteine for 5 min and then chased by the addition of excess of unlabeled methionine and cysteine. Invertase  was immunoprecipitated from labeled extract and resolved by  SDS-PAGE. position of core glycosylated er form and mature Golgi and secreted forms of invertase are indicated. immunolocalization of Lst1p-HA. CKY535 (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 antibody.  nuclear dna was visualized by dapi staining. Cell bodies were  visualized by differential interference contrast microscopy  dic). Bar, 1 mum. The intracellular distribution of Lst1p. (A) Cells expressing Lst1p-HA from a centromeric plasmid (CKY535) were  gently lysed and subjected to sequential centrifugation step, giving 500 g, 10,000 g, and 150,000 g pellet fraction (p) and 150,000 g supernatant fraction (s). Each sample contains extract  from the same number of cells. b cell lysate 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. Pellet (P) and supernatant  (S) fractions were then separated by centrifugation at 50,000 g.   lst1p-ha was detected by SDS-PAGE and immunoblotting  with anti-ha antibody. Two-Hybrid Interaction between LST1 and SEC23       beta-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 +- 5.3 Fusions to the LexA DNA-binding domain and to transcriptional activation domain  were induced by growth in galactose for 10 h. activities shown are mean from independent transformant. unit of beta-galactosidase activity are nmol/mg x min         lst1p/-sec23p complex is membrane associated. (A)  affinity isolation of lst1p/-sec23p complex. GST-Lst1p-HA  or GST alone was coexpressed with Sec23p and isolated by affinity to glutathione sepharose bead. Proteins bound to glutathione bead were loaded in lane 2 and 4. -sixth of the total lysate was loaded in lanes 1 and 3. (B) GST-LST1-HA,  SEC23, or both were expressed from the GAL1 promoter. cell   lysate were cleared of cell debris by centrifugation at 300 g for 2   minpellet p) and supernatant s fraction from cleared cell lysate were separated by centrifugation at 10,000 g for 30 min. aliquot of total cleared lysate t) was removed before centrifugation. An equal number of cell equivalents were loaded for  each sample. gst-lst1p-ha fusion was detected using anti-HA antibodies. For both A and B, sec23p protein was detected using anti-Sec23p antibodies. abbreviation used in paper dapi 4,6-diamidino-2-phenylindole gst glutathione s-transferase ha hemagglutinin epitope LST lethal  with sec-thirteen pma1p plasma membrane proton-atpase smm supplemented minimal medium ypd rich medium references topography of glycosylation in yeast: characterization of gdp mannose transport and lumenal guanosine diphosphatase activity in golgi-like vesicle cargo selection by copii budding machinery during export from the ER SEC12encodes guanine-nucleotide- exchange factor essential for transport vesicle budding from er copii: membrane coat formed by sec protein that drive vesicle budding from endoplasmic reticulum analyzing protein-protein interaction using -hybrid system coincident localization of secretory plasma membrane protein in organelle of yeast secretory pathway maturation of yeast plasma membrane  [H+]ATPase involves phosphorylation during intracellular transport targeting of the yeast plasma membrane  [H+]ATPase: novel gene ast1prevents mislocalization of mutant atpase to vacuole replacement of promoter of the yeast plasma membrane atpase gene by galactose-dependent promoter and physiological consequence functional complementation of null mutation of yeast saccharomyces cerevisiaeplasma membrane h(+)-atpase by plant h(+)-atpase gene lambda yes: multifunctional cdna expression vector for isolation of genes by complementation of yeast and Escherichia colimutations Genes that control fidelity of endoplasmic reticulum to Golgi transport identified as suppressor of vesicle budding mutation yeast sec16gene encodes multidomain vesicle coat protein that interacts with Sec23p SED4encodes yeast endoplasmic reticulum protein that binds Sec16p and participates in vesicle formation copii coat subunit interaction: Sec24p and Sec23p bind to adjacent region of sec16p fused protein domain inhibit dna binding by LexA Cdi1, human g1 s phase protein phosphatase that associates with cdk2 dominant lethal mutation in plasma membrane h+-atpase gene  of saccharomyces cerevisiae.  Putting the HO gene to work: practical use for mating-type switching sec23p and novel 105-kda protein function as multimeric complex to promote vesicle budding protein transport from endoplasmic reticulum vma21p is yeast membrane protein retained in the endoplasmic reticulum by di-lysine motif and is required for  the assembly of vacuolar h+-atpase complex vma22p is novel endoplasmic reticulum- associated protein required for assembly of yeast vacuolar h+-atpase complex vma12 encode yeast endoplasmic   reticulum protein required for vacuolar h+-atpase assembly yeast saccharomyces cerevisiaeselectable marker in pUC18 polylinker distinct set of secgene govern transport vesicle formation and fusion early in secretory pathway amino acid permease require COPII components and er resident membrane-protein shr3p for packaging into transport vesicles in vitro copii-cargo interaction direct protein sorting into er-derived transport vesicles classical mutagenesis techniques shr3: novel component of the secretory pathway specifically required for localization of amino acid permeases in yeast copii-coated vesicle formation reconstituted with purified coat protein and chemically-defined liposome pleiotropic plasma membrane atpase mutation of Saccharomyces cerevisiae.  Role of nad-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae.  determinant for  glycophospholipid anchoring of saccharomyces cerevisiaegas1 protein to the plasma membrane characterization of dominant lethal mutation in yeast   plasma-membrane h+-atpase gene growth control strength and active site of yeast plasma membrane ATPase studied by site-directed mutagenesis transport of axl2p depends on erv14p, er-vesicle protein related to the Drosophilacornichon gene product Immuno-fluorescence methods for yeast cytosolic sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro physiological regulation of  membrane protein sorting in the Golgi of Saccharomyces cerevisiae.  control of amino-acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7, and LST8.  Construction and use of gene fusion lacz(beta-galactosidase) which are expressed in yeast coat protein and vesicle budding yeast plasma membrane ATPase is essential for growth and has homology with (na+ + k+),  k+- and ca2+-ATPase copii subunit interaction in the assembly of the vesicle coat system of shuffle vector and yeast host   strain designed for efficient manipulation of DNA in Saccharomyces cerevisiae.  proliferation  of intracellular structure upon overexpression of pma2 atpase in Saccharomyces cerevisiae.  multisubunit complex associated with rna polymerase ii ctd and tata-binding protein in yeast comparison of the Saccharomyces  cerevisiaeG1 cyclins: Cln3 may be upstream activator of cln1, cln2, and cyclin physiology of mutants with reduced expression of plasma membrane h+-atpase functional expression of plant plasma membrane h(+)-atpase in yeast endoplasmic reticulum 