LST1 Is a SEC24 Homologue Used for Selective Export of the Plasma Membrane ATPase from the Endoplasmic Reticulum 
In Saccharomyces cerevisiae, vesicles that  carry proteins from the ER to the Golgi compartment  are encapsulated by COPII coat proteins. We identified  mutations in ten genes, designated LST (lethal with sec-thirteen), that were lethal in combination with the COPII  mutation sec13-1. LST1 showed synthetic-lethal interactions with the complete set of COPII genes, indicating  that LST1 encodes a new COPII function. LST1 codes  for a protein similar in sequence to the COPII subunit  Sec24p. Like Sec24p, Lst1p is a peripheral ER membrane protein that binds to the COPII subunit Sec23p.  Chromosomal 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. Localization by both immunofluorescence  microscopy and cell fractionation shows that the export  of Pma1p from the ER is impaired in lst1Delta mutants.  Transport of other proteins from the ER was not affected by lst1Delta, nor was Pma1p transport found to be  particularly sensitive to other COPII defects. Together,  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.
The 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). The  proton gradient generated by Pma1p then drives the uptake of nutrients, such as amino acids, from the extracellular medium (Vallejo and Serrano, 1989). A second physiological function of Pma1p is to maintain the cytosol at a  neutral pH. In medium of low pH, the 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 the secretory pathway defined  by the sec genes (Brada and Schekman, 1988; Chang and  Slayman, 1991). Pma1p is one of the most abundant cargo  molecules of the secretory pathway, constituting 25-50%  of the total plasma membrane protein (Serrano, 1991). Because 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. Such 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). For early steps in the 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 a set of proteins known as COPII (Barlowe et  al., 1994). These 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). The stepwise recruitment and assembly of  the COPII coat onto the membrane is thought to occur as  follows. Action 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). Membrane-associated Sar1p, in turn, recruits the soluble Sec23p/Sec24p and Sec13p/Sec31p complexes (Matsuoka et al., 1998). Sec16p 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). To 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. Association 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).
An early indication that the COPII coat subunits would  physically interact came from specific genetic interactions  between mutations in COPII genes. When 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. 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).  The 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.
We screened for mutations that were lethal with the  COPII mutation sec13-1 and identified ten LST genes (lethal with sec-thirteen). As 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). Accordingly, these LST genes display synthetic-lethal interactions with SEC13, but not with the other COPII genes.  On 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. Here 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. The 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. These results suggest a specialized form of the vesicle coat that is responsible for recruitment of Pma1p into COPII-coated vesicles.
Materials and Methods
Media, Strains, and Plasmids
The Saccharomyces cerevisiae strains used in this 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 a yeast culture). For some experiments, SMM  was buffered to pH 6.5 using 50 mM MOPS and 50 mM MES. Genetic manipulations were performed according to standard protocols (Kaiser et al.,  1994). DNA manipulations were carried out as described in Sambrook  et al. (1989). pAF70 carries the SEC24 gene in the centromere vector  pCT3 (URA3; Gimeno et al., 1996). pKR34 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. pKR17 carries  the LST1 gene on a 3.5-kb fragment in the centromere vector pRS316  (URA3). A 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. To study the toxic  effects of LST1, pKR35 was constructed which contains the entire LST1  coding sequence expressed from pGAL1 on pCD43 (URA3). pKR35 will  prevent growth under conditions of full induction on galactose medium,  establishing that overexpression of Lst1p is toxic to yeast cells. Under conditions of partial induction of pGAL1-LST1 in cells 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, 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. pKR17HA 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.  Restriction analysis using sites flanking the point of insertion revealed  that two 100-bp inserts (six HA epitopes) were present in pKR17HA.  pKR17HA was transformed into CKY536 to make CKY535 (MATa lst1Delta::  LEU2 leu2-3, 112 ura3-52 [pKR17HA]).
Synthetic-lethal Screen
The following plasmids and strains were constructed for use in the sec13-1  synthetic-lethal screen. The 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). pKR4 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). CUY563 and CKY45 were crossed to produce a MATa ade2 ade3  leu2 ura3 sec13-1 segregant, which was transformed with pKR4 to give  CKY423. The mating type of CKY423 was switched by ectopic expression  of the HO gene (Herskowitz and Jensen, 1991) to give CKY424.
Cultures of CKY423 and CKY424 were mutagenized by irradiation  with a germicidal UV lamp at a dose resulting in 10% cell survival. Mutagenized cells were plated on YPD at a density of 150 colonies per plate.  After 5 d of growth at 24 C, colonies with a solid red color and no white  sectors were selected for further analysis. The dependence of the nonsectoring phenotype on the sec13-1 mutation was tested by transforming candidate mutants with either pKR1 or pRS316. Strains that sectored after  transformation with pKR1, but not after transformation with pRS316,  were scored as sec13-1-dependent.
Complementation tests were performed by mating mutants isolated  from CKY423 with those isolated from CKY424. Zygotes isolated by micromanipulation were scored for their ability to form sectored colonies on  YPD plates. The genes defined by these complementation groups were  designated LST. All lst mutant strains were backcrossed to a parental  strain twice.
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, using the integrating plasmid p1312 (SEC13 URA3; Pryer et al., 1993). The integrants  were grown on YPD and cells from white sectors (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, a  temperature that is restrictive for the sec13-1 mutation. Owing to the poor  growth of lst9 strains, we were not able to construct a lst9 single mutant by  this method.
To 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). Inviability 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. The 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.
Construction of lst1Delta Mutants
Replacement of the chromosomal LST1 gene with an allele disrupted with  the LEU2 gene was constructed as follows. pKR18 carries the 5' half of  LST1 on a 2.0-kb Xho1/HindIII fragment inserted into pRS316. A 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.  The 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. The 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). On sporulation  and dissection, this diploid gave four viable spore clones, and haploid segregants carrying lst1Delta::LEU2 were confirmed by Southern blotting. One  such segregant was further backcrossed to our S288C genetic background  to give strains CKY536 and CKY542.
Proton Efflux from Intact Yeast Cells
Pma1p activity was assayed by proton efflux from intact cells into the 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 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. The  pH of the 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 a final concentration of 40 mM and the ensuing drop in  pH was recorded at 30-s intervals over 15 min. In 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.
Immunofluorescence Microscopy
The 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). Strains were grown exponentially in SMM medium, pH  7.2, at 30 C. Cells were fixed in 3.7% formaldehyde and then converted to  spheroplasts by digestion with lyticase. Both primary and secondary antibody incubations were for 1 h at 25 C. Affinity-purified anti-Pma1p antibody was prepared as follows. A 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. Rabbit 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. Affinity-purified Pma1p was used at a 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 a  Nikon Eclipse TE300 microscope with a Hamamatsu Orca C4742-95 CCD  camera.
For the localization of Lst1p-HA, CKY535 was grown on SMM to exponential phase and prepared as described above. For 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. Rabbit 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.  Samples were viewed and imaged using a Nikon Optiphot 2 microscope  and a Photometric ImagePoint CCD camera. Images were recorded using  IP-Lab software (Molecular Dynamics, Inc.).
Cell Fractionation
Cell organelles were fractionated on equilibrium density gradients 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 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. An 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. The 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.). Samples were centrifuged 100,000 g for 18 h at 4 C  and fractions of 300 mul were collected from the top of the gradient. Protein  was precipitated from each fraction by the addition of 100 mul of 0.15%  deoxycholate and 100 mul of 72% trichloroacetic acid. Protein 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). Pma1p, Gas1p, and Sec61p were  resolved by SDS-PAGE and were detected by immunoblotting. The relative amount of each protein in cell fractions was determined by densitometry using an Ultroscan 2202 (LKB Instruments, Inc.). The Golgi GDPase  activity was assayed in gradient fractions before protein precipitation using standard methods (Abeijon et al., 1989).
The 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 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. The 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.
Release 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. 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
Samples 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 the transfer buffer. The 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. Blots were developed using chemiluminescence detection system (Nycomed Amersham Corp.).
Pulse-Chase Kinetics of Invertase Maturation
The 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). Wild-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. A 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).
Two-Hybrid Interactions
The yeast two-hybrid assay was used to test potential protein-protein interactions as previously described (Gyuris et al., 1993; Bartel and Fields,  1995). Interactions were tested between either Lst1p or Sec24p fused to  the LexA DNA-binding domain and Sec23p fused to an acidic transcriptional activation domain. The 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).
Combinations of control and fusion protein plasmids, along with the reporter plasmid pSH18-34, were transformed into the strain EGY40 (Golemis and Brent, 1992). Strains were grown exponentially in SMM with 2%  raffinose as the carbon source. Galactose was added to a concentration of  2%, and incubation was continued for 10 h to induce fusion proteins expressed from pGAL1. Assays 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
A 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. pPE123  is the SEC23 gene expressed from pGAL1 in pRS315 (Gimeno et al., 1996).  Binding interactions were tested from extracts 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 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. The extract was diluted to 1 ml with IP buffer, and membranes were collected by centrifugation at 500 g for 20 min. This 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. After 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.). The 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. Proteins were released  from glutathione Sepharose 4B beads by solubilization in ESB. Samples  of total lysate were prepared by adding 2x ESB to an equal amount of the  diluted extract from the salt washed membranes. Samples were analyzed  by immunoblots probed with anti-Sec23p antibody.
For 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. 2 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. 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 cells and large cell debris by centrifugation at 300 g for 3 min. 50 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. An equal number of cell equivalents of total extract, membrane-pellet, and supernatant fractions was solubilized in ESB and analyzed  by immunoblotting. The cytosolic protein Gdh2p was found only in the soluble fractions, demonstrating cell lysis was complete (data not shown).
Results
Mutations Synthetically Lethal with sec13-1
To 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). Strain CKY423 has  the chromosomal mutations ade2 ade3 sec13-1 and harbors  the plasmid pKR4, which carries wild-type copies of  SEC13 and ADE3. This 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. In this strain, a mutation that is lethal with sec13-1 will produce a nonsectoring colony. Mutagenesis of CKY423 and the isogenic strain of opposite  mating type, CKY424, yielded 139 nonsectoring mutants  (Fig. 1). These strains were then tested for restored ability  to sector after transformation with pKR1, which carries  wild-type SEC13, but lacks the ADE3 gene. By this test, 57  of the mutants had synthetic-lethal mutations that could  be rescued by wild-type SEC13. In backcrosses, 52 mutants gave a segregation pattern indicating that the trait  was due to a single nuclear mutation (Fig. 1). 
Matings between mutants identified 11 complementation groups using colony sectoring of the diploid as the criterion for allelic complementation. These complementation groups were designated LST (Table II). One of the  complementation groups was shown to comprise recessive  lethal mutations in the SEC13 gene itself (Roberg et al.,  1997b). Tests for rescue of the nonsectoring phenotype by  plasmids carrying known sec genes showed that LST10  was SEC16 (Roberg et al., 1997b). 
Synthetic Interactions of lst Mutations
To 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). Representative lst  single mutants were then crossed to sec16, sec23, and sec31  mutants. 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 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). Mutations in LST1 were inviable when combined with sec16, sec23, and sec31 mutations, 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 crosses 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 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. 
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 the presence of the lst1-1 mutation.  CKY426 was transformed with yeast genomic libraries. 34  colonies that regained the ability to form white sectors  were identified among 97,000 Ura+ transformants. We expected this screen to yield plasmids carrying either SEC13  or LST1. About 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. The restriction maps of the remaining rescuing  plasmids show that they represent two unrelated chromosomal regions. The clones p21-31 and p77-2 were selected  as representatives of each region. The 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. For chromosomal  integration, pKR20 was linearized by digestion with HpaI  and transformed into CUY564 (MATalpha ade2 ade3 leu2  ura3). The resulting strain was crossed to the lst1-1 mutant  CKY426 (MATa lst1-1 sec13-1 ade2 ade3 leu2 ura3  [pKR4]). After 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-.  Thus, p77-2 carries the LST1 gene. In 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. pKR7 was integrated at its 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 an unlinked suppressor gene.
The 3.5-kb insert of p77-2 was inserted into the XhoI  site of the centromeric vector pRS316 to construct pKR17.  The base sequence of this insert was determined and  found to contain a single open reading frame encoding a  protein of 929 amino acids. This sequence corresponds to  the open reading frame YHR098c located on chromosome  VIII (Saccharomyces Genome Database, Cherry et al.,  1997). The predicted amino acid sequence of LST1 shows  significant similarity to SEC24 (YIL109C). The 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. 
Phenotypes of lst1Delta
One copy of the LST1 gene in the wild-type diploid strain  CKY348 was disrupted to generate a lst1Delta::LEU2/LST1  heterozygote. Sporulation 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. A lst1Delta::LEU2 mutant spore clone was  crossed to sec mutants to test for synthetic lethality. In  these crosses, both the temperature sensitivity of the sec  mutation and the lst1Delta allele marked by LEU2 could be  followed independently. In 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). Tests of the genotype  of the surviving sister spore clones showed that the inviable spores in these crosses were always lst1Delta sec double  mutants. Crosses between lst1Delta and sec17 or sec18 produced viable double mutants. These 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.
We evaluated the growth of lst1Delta::LEU2 mutants under  a variety of conditions. On YPD, the 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 an acidic environment, and we  tested the 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  mutants 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 conditions. 
Having identified conditions where LST1 was needed  for growth, we investigated whether overexpression of  SEC24 could supply the function lost in lst1Delta. Some 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). These findings imply some functional overlap  between LST1 and SEC24. In 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. We 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. By 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). 
In 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. To 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. A 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. When 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). 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 the 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 an identical halo assay, and were found to be resistant to  the effect of galactose (Fig. 3 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  a 2mu plasmid significantly slows the growth of our yeast  strains. Any suppression afforded by overexpression of  SEC23 might be counteracted by this inherent toxicity of  SEC23. A simple conclusion that can be drawn from these  overexpression studies is that too great of a stoichiometric  excess of Lst1p over Sec24p is lethal. This 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.
lst1Delta Diminishes the Activity of the Plasma Membrane Proton-ATPase
The 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). The dependence of Pma1p activity on LST1 was supported by the  observation that lst1Delta mutants exhibited an unusual morphology characteristic of pma1 mutants. 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 rosettes; in some cases, as many as 15 daughters radiated  from a single large mother cell (Fig. 4 A). The 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. Cells depleted of Pma1p produce  similar multibudded cells with attached daughters that had  completed cytokinesis. In 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). The 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. 
As 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. Wild-type and  lst1Delta strains 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  a weakly buffered medium and proton efflux on addition  of glucose was measured as a drop in extracellular pH. For  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). Although 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.  These findings indicate that the lst1Delta mutant grown at 37 C  has about half of the Pma1p activity as wild-type cells.
LST1 Is Required for Efficient Transport of Pma1p Out of the ER
To 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. Cells were grown at 30 C in YPD medium to  avoid possible secondary effects due to the pH sensitivity  of lst1Delta mutants. In 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). This 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).
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 gradients under conditions where the ER and plasma membrane are well separated on the basis of their buoyant density. Pma1p 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). In 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).  Interestingly, 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. Given 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. A 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). 
The 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.  The 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. As 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. No 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 the ER form to the  Golgi and vacuolar forms of the enzyme could be detected  (data not shown). 
We also considered the possibility that transport of  Pma1p may be particularly sensitive to any subtle defect in  vesicle formation. We addressed this possibility by examining the localization of Pma1p in sec24-1 and sec31-2 mutant cells at the 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 the perinuclear  region of either mutant by immunofluorescence microscopy (data not shown). Thus, partial defects in COPII  functions did not lead to the extensive accumulation of  Pma1p in the ER that was observed for lst1Delta mutants.  Taken 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.
Localization of Lst1p
To 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. The 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). In cells expressing  Lst1p-HA that were fixed for immunofluorescence microscopy, staining was found primarily at the nuclear periphery (Fig. 8). No signal was seen in cells expressing untagged Lst1p, verifying that the origin of the staining  pattern was due to Lst1p-HA. Although 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. In addition, weak  punctate staining was observed throughout the cell body,  some of which may correspond to ER membranes near the  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.  This 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 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). The association of the Lst1p protein  with the 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). The 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. 
Lst1p Binds Sec23p
Sec24p was first identified as a protein that formed a 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 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). Interaction  between the two fusion proteins was tested by assaying for  activation of a lacZ reporter gene. Induction of beta-galactosidase was observed when the LST1 and SEC23 fusions  were coexpressed, but not when expressed alone (Table  IV). The 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 extracts. The coding sequence of LST-1-HA (codons 14-927)  was fused to GST and expressed in yeast from the pGAL1  promoter. SEC23 was also expressed from pGAL1. Since  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. GST-Lst1p-HA was  isolated from the extracts by affinity to glutathione  Sepharose beads. Sec23p was found in association with  GST-Lst1p-Ha, but not in control extracts 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 an 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 the 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 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). 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 a 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 the 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 the Lst1p/ Sec23p complex has affinity for ER membranes.
Discussion
By screening for mutants that exhibited synthetic-lethal  genetic interactions with the COPII mutation sec13-1, we  identified the LST1 gene. Subsequent 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). This 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. The 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. The  two proteins share 23% amino acid identity over their entire lengths. (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 two-hybrid interaction and affinity purification of a complex of GST-Lst1p and Sec23p.  (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. (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.  Based 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.
Strains 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. 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, 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. Therefore, it seems likely that  Lst1p and Sec24p share the 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.
The 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 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. This 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.
We also considered the possibility that there may be differences among cargo molecules in their response to general defects in the 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 our standard marker proteins might have a significant  effect on the rate of transport of Pma1p. If this were the  case, partial defects in other COPII components should  also interfere with Pma1p transport. Therefore, 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. Although 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.
Factors required for the transport of specific membrane  proteins have been documented in a number of other  cases. The 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). A 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). Similarly, 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). 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, 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. Because 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.
Expression 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). Similarly, 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). One 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). The 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. In  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.
The 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. Using  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. In 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). The 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.  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 vesicles.
We 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. Soluble Lst1p-HA  gives a high background in vesicle fractions preventing us  from reliably determining whether there is a specific association of Lst1p with vesicles. In 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. It 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. Finally, it will be of interest to determine if there is direct binding of Lst1p to  Pma1p.
The 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. It is  possible that Sec23p/Lst1p complexes act to form a class of  vesicle that is distinct from those formed by Sec23p/ Sec24p complexes. Alternatively, it is possible that the two  complexes assemble together forming vesicles with coats  of mixed composition. The identification of additional homologues of Sec23p and Sec24p suggest the existence of  coats with even greater combinatorial complexity. We  have identified a third 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, the  Saccharomyces genome contains an 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  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.
Figures 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 a red colony. Mutagenized cells that have acquired an lst  mutation cannot grow without the pKR4 plasmid and form nonsectoring, solid red colonies. Of 132 nonsectoring colonies, the  sectoring in 57 was restored by transformation with a 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 sequences. Identities  are indicated by solid lines and  similarities are indicated by dotted lines. Overall amino acid  identity is 23%.
Functional relationships 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) 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. Colonies were photographed after growth at 37 C for 2 d. (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). Transformants  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. 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. The same fields of cells,  stained with DAPI to label the nuclear  DNA, are also shown. Top 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). Bar, 5 mum.
Pma1p defects  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 this type comprise  ~10% of a 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 a decrease in the pH of the external medium. Based 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.
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 lysates were fractionated on density gradients of 20-60% sucrose. Relative levels 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 the 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 the 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 an excess of unlabeled methionine and cysteine. Invertase  was immunoprecipitated from labeled extracts and resolved by  SDS-PAGE. Positions of the 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 antibodies.  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 steps, giving 500 g, 10,000 g, and 150,000 g pellet fractions (P) and a  150,000 g supernatant fraction (S). Each sample contains extract  from the same number of cells. (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. 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 a transcriptional activation domain  were induced by growth in galactose for 10 h. Activities shown are the mean from five  independent transformants. Units of beta-galactosidase activity are nmol/mg x min.       
Lst1p/-Sec23p complex is membrane associated. (A)  Affinity isolation of Lst1p/-Sec23p complexes. GST-Lst1p-HA  or GST alone was coexpressed with Sec23p and isolated by affinity to glutathione Sepharose beads. Proteins bound to glutathione beads were loaded in lanes 2 and 4. One-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  lysates were cleared of cell debris by centrifugation at 300 g for 2  min. Pellet (P) and supernatant (S) fractions from cleared cell lysates were separated by centrifugation at 10,000 g for 30 min. An  aliquot of the total cleared lysate (T) was removed before centrifugation. An equal number of cell equivalents were loaded for  each sample. The GST-Lst1p-HA fusion was detected using anti-HA antibodies. For both A and B, the Sec23p protein was detected using anti-Sec23p antibodies.
Abbreviations used in this 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
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