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© The Rockefeller University Press, 0021-9525/1998//1209 $5.00
The Journal of Cell Biology, Volume 142, Number 5, , 1998 1209-1222

Transport of Axl2p Depends on Erv14p, an ER–Vesicle Protein Related to the Drosophila cornichon Gene Product

Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755

COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides. One of these polypeptides, termed Erv14p (ER–vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product. Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles. Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not efficiently delivered to the cell surface. Axl2p is required for selection of axial growth sites and normally localizes to nascent bud tips or the mother bud neck. In erv14 strains, Axl2p accumulates in the ER while other secretory proteins are transported at wild-type rates. We propose that Erv14p is required for the export of specific secretory cargo from the ER. The polarity defect of erv14 yeast cells is reminiscent of cornichon mutants, in which egg chambers fail to establish proper asymmetry during early stages of oogenesis. These results suggest an unforeseen conservation in mechanisms producing cell polarity shared between yeast and Drosophila.

Key Words: ER • Golgi • vesicles • coat proteins • cell polarity

Abbreviations used in this paper: CPY, carboxypeptidase Y; Endo H, endoglycosidase H; Erv, ER vesicle; HA, hemagglutinin; PMA, plasma membrane ATPase.

SECRETORY proteins destined for intracellular organelles or the plasma membrane are first synthesized and processed at the ER of eukaryotic cells. Fully folded secretory proteins are then packaged into ER-derived transport vesicles for export to the Golgi complex and beyond. Several lines of experimental evidence indicate mechanisms of protein retention and retrieval operate during ER–Golgi transport to maintain distinct organelle identity (Sato et al., 1996; Kaiser et al., 1997). In addition, secretory proteins that must move forward are concentrated into ER-derived vesicles during export from this compartment (Quinn et al., 1984; Balch et al., 1994; Rexach et al., 1994); however, the mechanisms of this selection procedure remain obscure. One component of this selective export process is a protein complex, termed COPII, that forms ER-derived transport vesicles and selects secretory proteins by direct or indirect interaction (Barlowe et al., 1994; Kuehn et al., 1998). In the yeast Saccharomyces cerevisiae, formation of ER-derived vesicles has been reconstituted in a cell-free reaction with ER membranes and purified COPII proteins (Salama et al., 1993). We have proposed that additional components of this selection machinery are contained on ER-derived vesicles, and we have undertaken a molecular analysis of protein constituents on purified COPII-coated vesicles (Belden and Barlowe, 1996).

An uncoated form of ER-derived vesicles may be isolated after centrifugation on density gradients. These gradient-purified vesicles contain a set of tightly associated polypeptides that are solubilized by detergents but not by an elevated pH treatment. NH₂-terminal polypeptide sequences have been determined from several of the abundant species contained on ER vesicles (Ervs),¹ starting with the lowest molecular weight species moving upward (Belden and Barlowe, 1996). In this report, we characterize Erv14p, an integral membrane protein that localizes to the ER and Golgi compartments of yeast cells. Strains that lack Erv14p (erv14) are viable but display novel phenotypes in both haploid and diploid stages of growth. First, haploid erv14 strains are defective in selecting the proper bud site. Wild-type budding yeast are highly polarized during vegetative growth, orienting cytoskeletal elements and the secretory pathway toward the emerging bud tip (Drubin et al., 1991). Haploids select bud sites such that mother and daughter cells bud toward each other and therefore exhibit an "axial" budding pattern. Genetic analyses in yeast have led to the identification of several genes that are required for selection of axial growth sites (Chant, 1996). One of these gene products, Axl2p, is an integral membrane secretory protein that must be delivered to the plasma membrane for establishment of the axial bud site (Halme et al., 1996; Roemer et al., 1996). In erv14 strains, Axl2p is largely retained in the ER and the yeast bud in a nonaxial manner. The accumulation of Axl2p in the ER appears to be selective since other secretory proteins examined were transported at wild-type rates. Second, diploid erv14/erv14 strains display normal diploid budding patterns but do not sporulate when deprived of nutrients. This sporulation defect does not appear to be related to transport of Axl2p and suggests that Erv14p participates in the transport of additional secretory proteins.

Erv14p shares a high degree of amino acid identity (36%) with the cornichon gene product from Drosophila melanogaster. Mutations in the cornichon gene are known to disrupt anterior–posterior pattern formation during early stages of Drosophila oogenesis and ultimately lead to misorientation of the oocyte cytoskeleton (Roth et al., 1995). This phenotype is in some respects similar to the erv14 phenotype. Therefore, experiments with the yeast homologue of cornichon may provide insight on the molecular mechanisms establishing polarity in the Drosophila oocyte and perhaps in a variety of cell types.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Yeast Strains and Media
The strains used in this study are listed in Table I. Cultures were grown in either rich medium (1% Bacto-yeast extract, 2% Bacto-peptone, and 2% dextrose [YPD]) or minimal medium (0.67% nitrogen base without amino acids, 2% dextrose [YMD]) containing appropriate supplements. Cultures were grown at 30°C unless indicated differently in figure legends. Standard yeast genetic methods used in these studies have been previously described (Sherman, 1991). Manipulation of recombinant DNA was performed as described (Ausubel et al., 1987), and Escherichia coli strain DH5 (Woodcock et al., 1989) was used for these procedures.

Strain Construction
The ERV14 locus was targeted for disruption with the HIS3 gene (Baudin et al., 1993). A PCR method was used to amplify an erv14::HIS3 disruption fragment using the primers GP1 (5'-TGCAATTAAAGTAAAGTAAAAAAATTAAGAATAAAAAGAAAAGGCCTCCTCTAGTACACT-3') and GP2 (5'-CTTGGCCCTTCAGTCTTCTTTGGATTTC- AATGTCTTGTTGGAGCGCGCCTCGTTCAGAATG-3') with pHISKO as a template. The resulting product has the HIS3 gene flanked by 45 bp of the ERV14 gene directly before the ATG start and directly after the stop codon. Transformation of strain YPH501 (Sikorski and Heiter, 1989) to histidine prototrophy and subsequent screening identified several isolates that were heterozygous at the ERV14 locus. Several of these heterozygous diploids were grown under conditions to induce sporulation, and dissection of these asci produced four viable spores. A haploid disruptant (CBY251) was also constructed in strain FY834 and used in subsequent studies. Disruption of the ERV14 locus was confirmed by two independent PCR analyses. First, the GP3 primer and a primer internal to the HIS3 gene (5'-GCCTCATCCAAAGGCGC-3') generated a product (650 bp) of the expected size, and second, primers that flank the ERV14 gene (GP3 and GP4) produced a single product (1,500 bp) of the expected size.

An Erv14p homologue that shares 63% amino acid identity is defined by open reading frame YBR210w on chromosome II (Feldmann et al., 1994), referred to here as ERV15, and was also targeted for disruption with the TRP1 gene in a multistep procedure. The primers EF4 (5'-AATCTAGAGCTCACTACTACTCTCTC-3') and EF3 (5'-AAACTCGAGCAAATACGAGGGAGATCG-3'), which correspond to regions that are 210 bp upstream and 300 bp downstream of the open reading frame, were used to amplify ERV15 from genomic DNA. These primers contain the restriction sites XbaI (EF4) and XhoI (EF3) to facilitate insertion into the homologous sites of pRS305 (Sikorski and Heiter, 1989). This plasmid, pREF305, was modified to disrupt the open reading frame of ERV15 with TRP1. A unique PstI site located 29 bases upstream of the ERV14 start codon and a partial digest of DraI (site is located 328 bp downstream from the ATG start) were used to insert the TRP1 gene. A PstI/SmaI digest of pJJ248 (Jones and Prakash, 1990) liberates a 955-bp fragment containing the TRP1 gene that was then inserted into pREF305 treated with PstI and DraI. The resulting plasmid, pEFT305, was digested with NotI/XboI, and the 1.6-kbp fragment containing the TRP1 gene flanked by ERV15 sequence was isolated. This disruption fragment was used to transform strain FY833. Tryptophan prototrophs were screened using the primers EF7, 5'-CTGAACGACAAAGTGAAGC-3' (anneals 404 bp downstream of the ERV15 stop codon) and JP7, 5'-TCACCTGTCCCACCTGC-3' (anneals 500 bp downstream from the TRP1 start codon), which produce a 1.1-kbp amplification product if integration has occurred at the ERV15 locus. One of these isolates, CBY347, was used in subsequent experiments and was mated with CBY251 to generate the double erv14::HIS3 erv15::TRP1 strain, CBY348. This strain was placed in media to induce sporulation, and an isogenic set of strains containing the single and double disruptions (CBY353, CBY354, CBY355, and CBY356) were isolated for use in these studies. To prepare diploids homozygous for erv14::HIS3, CBY356 was mated with CBY358, and zygotes were picked by their distinctive morphology under the microscope.

Antibodies and Immunoblotting
Antibodies directed against Sec61p (Stirling et al., 1992), Sec22p (Bednarek et al., 1995), Erv25p (Belden and Barlowe, 1996), Emp47p (Schröder et al., 1995), Sec23p (Hicke and Schekman, 1989), Kar2p (Brodsky and Schekman, 1993), CPY (Rothblatt et al., 1989), Gas1p (Frankhauser and Conzelmann, 1991), Vph1 (Kane et al., 1992), and plasma membrane ATPase (Carolyn Slayman, Yale University, New Haven, CT) were used in these studies. Anti-HA monoclonal antibody (HA.11) raised against the peptide CYPYDVPDYASL was obtained from Berkeley Antibody Co. (Richmond, CA). Polyclonal antiserum specific for Sec12p was raised against the NH₂-terminal domain of Sec12p (amino acid residues 1–354) fused to protein A. Fusion protein was expressed from the plasmid pRIT33 (Nilsson and Abrahmsen, 1990) and purified by IgG affinity chromatography as described by the manufacturer (Pharmacia Biotech, Piscataway, NJ). Antigen (0.25 mg) was mixed with Freund's complete adjuvant and injected into rabbits followed by monthly boosts with 0.12 mg of antigen in Freund's incomplete adjuvant. Serum isolated from immunized rabbits cross-reacted with a 70-kD species that was overproduced when transformed with a GAL1-regulated version of SEC12 (d'Enfert et al., 1991). For immunoblots, proteins were resolved by SDS-PAGE (Laemmli, 1970) and transferred to nitrocellulose (Towbin et al., 1979), and filter-bound primary antibodies were detected by peroxidase-catalyzed chemiluminescence (ECL method; Amersham Corp., Arlington Heights, IL).

In Vitro Vesicle Budding
Microsomes were isolated from strain CBY409 and incubated in the presence or absence of proteins required for reconstitution of vesicle formation as described (Barlowe et al., 1994). A 15-µl portion of the total reaction and 150 µl of a supernatant fluid containing vesicles released from 200-µl budding reactions were centrifuged at 100,000 g (model TLA100.3 rotor; Beckman Coulter, Inc., Fullerton, CA) to collect membranes. The pellet fractions were dissolved in 30 µl of SDS-PAGE buffer, and 7–10 µl of this material was resolved on 12.5% polyacrylamide gel and immunoblotted for Sec22p, Erv25p, Sec12p, and anti-HA for Erv14p-HA detection.

Subcellular Fractionation
Subcellular fractionation was performed as described by Antebi and Fink (1992) with modifications by Schimmöller et al. (1995). Strains were grown to exponential phase and converted to spheroplasts by lyticase treatment (Baker et al., 1988). Spheroplasts were centrifuged and resuspended in a sucrose solution (10 mM Hepes, pH 7.5, 12.5% sucrose, 1 mM EDTA, 1 mM PMSF) and subjected to 10 strokes in a Dounce homogenizer. Two clearing spins were performed, and the resulting supernatant fluid was placed on a sucrose gradient consisting of nine steps from 22 to 60% (wt/vol) in 10 mM Hepes, pH 7.4, 1 mM MgCl₂. The gradients were centrifuged at 35,000 rpm (model SW40 rotor; Beckman Instruments, Palo Alto, CA) for 2.5 h at 4°C. 15 fractions of 0.77 ml each were taken sequentially from the top of the gradient to the bottom. Fractions were diluted in SDS-PAGE sample buffer, and proteins were resolved on polyacrylamide gels and immunoblotted for Sec61p (ER marker), Emp47p (Golgi marker), plasma membrane ATPase (PMA), Vph1 (vacuolar marker), and anti-HA monoclonal antibody to detect Erv14p-HA or Axl2-HA. Relative levels of specific proteins in each fraction were quantified by densitometry of immunoblots. GDPase activity was determined as described (Yanagisawa et al., 1990) using CDP to subtract nonspecific phosphatase activity. Sucrose concentrations of individual fractions were determined by measuring the refractive index with an Abbe Refractormeter (American Optical, Buffalo, NY).

For subcellular fractionation under conditions of divalent cation chelation, the method of Kölling and Hollenberg (1994) was followed. Cells were grown and spheroplasted as described above; however, lysates were prepared by agitation with glass beads in buffer containing 10 mM Hepes, pH 7.5, 12.5% sucrose, 10 mM EDTA, 1 mM PMSF. The cleared lysate was loaded onto a similar 22–60% sucrose gradient as described above, except gradients contained 10 mM EDTA, and samples were centrifuged at 30,000 rpm (model SW40 rotor; Beckman Instruments) for 14 h at 4°C. Fractions were collected and analyzed as described above for magnesium-containing gradients.

To characterize the membrane association of Erv14p, yeast cell membranes were isolated and treated with various agents as follows. Spheroplasts were prepared as indicated above and resuspended in buffer 88 (20 mM Hepes, pH 6.8, 250 mM sorbitol, 150 mM KOAc, 5 mM MgOAc) containing 1 mM DTT and 1 mM PMSF. After Dounce homogenization, a low-speed supernatant fraction was prepared by centrifugation at 5,000 rpm (model SS34 rotor; Sorvall, Newtown, CT). Aliquots from the supernatant fraction were treated with buffer 88, 0.5 M NaCl, 2.5 M urea, 0.1 M sodium carbonate, or 1% Triton-X 100 in buffer 88. These samples were mixed and incubated 20 min on ice followed by centrifugation at 60,000 rpm (model TLA 100.3 rotor; Beckman Instruments) for 15 min. Equivalent amounts of supernatant and pellet fractions were diluted in SDS-PAGE buffer and resolved on a 12.5% polyacrylamide gel. Blots were probed with anti-Emp47p (integral membrane protein), anti-Sec23p serum (peripheral membrane protein), or anti-HA monoclonal antibody to detect Erv14p-HA.

Calcofluor Staining of Bud Scars
Yeast strains in a logarithmic stage of growth were diluted into YPD medium to an OD₆₀₀ of 0.002. When cultures reached an OD₆₀₀ of 0.5–0.8, an aliquot (1.5 ml) of cells was harvested and resuspended in calcofluor stain at a concentration of 0.1 mg/ml, as described (Pringle, 1991). After incubation for 5–10 min at room temperature, the cells were washed three times in distilled water and resuspended in a final volume of 50 µl. Stained yeast were viewed under a fluorescence microscope, and cells possessing six or more bud scars were examined for an axial or nonaxial budding pattern. Haploid cells that displayed one or more bud scars at opposite poles were scored as nonaxial.

Sporulation and Cell Survival Experiments
Sporulation efficiency and cell survival studies were performed as follows. Diploid strains were cultured for 8 h at 30°C in YPD to an exponential phase of growth. Cells were then harvested, washed twice with water, and resuspended to an OD₆₀₀ of 1 in spmD, a nitrogen-deficient medium composed of 1% potassium acetate, 0.1% yeast extract, and 0.05% dextrose. To assess sporulation efficiency, samples from these cultures were microscopically examined daily to determine the number of tetrads per 300 cells. Cell viability from these cultures was assessed over a 7-d period by spreading an equivalent amount of OD₆₀₀ units on YPD plates. After 4 d of growth on YPD plates, the colonies were counted to determine cell viability.

Indirect Immunofluorescence
Yeast strains were grown in 25 ml of YPD to an OD₆₀₀ of 0.3 and fixed with 5% formaldehyde for 1 h. Fixed cells were then centrifuged for 3 min in a clinical centrifuge, washed three times in PBSS (PBS, pH 7.4, and 0.7 M sorbitol) and resuspended in a final volume of 400 µl PBSS. β-Mercaptoethanol (20 mM final) and lyticase were added and incubated at room temperature for 30 min to digest cell walls. The fixed spheroplasts were washed twice and resuspended in 400 µl of PBSS, and 15 µl was applied to polylysine-coated multiwell slides (Pringle et al., 1991). Cells were adhered for 10 min, washed, and incubated with blocking buffer (PBSS with 1% BSA and 0.5% Triton X-100) for 10 min. Wells were washed twice with PBSS followed by the addition of primary antibodies (anti-Kar2p at 1:500 and anti-HA at 1:200) diluted in blocking buffer. After incubation at room temperature for 90 min, cells were washed five times with PBSS and then incubated with secondary antibodies that had been diluted in blocking buffer at 1:500 (anti–rabbit IgG conjugated to fluorescein or anti– mouse IgG conjugated to Texas red). After 90 min, cells were washed four times with PBSS followed by a 1-min incubation in PBSS containing 1 µg/ ml diamidophenylindole. Cells were then washed four times with PBSS, and mounting medium (FITC guard; Testog, Inc., Chicago, IL) was layered on wells before sealing with coverslips. For all fluorescence microscopy, images were obtained using a cooled CCD camera, and composites were prepared with Adobe Photoshop (San Jose, CA) software.

In Vivo Labeling
Pulse-chase experiments were performed as previously described (Belden and Barlowe, 1996). In brief, cells were grown at 30°C (25°C for the sec12-4 strain) in selective medium containing 2% dextrose to an OD₆₀₀ of 0.5. Cultures were harvested, washed, and resuspended at one-tenth the original volume in selective medium lacking sulfate. After culturing for 10 min at 30°C (37°C for sec12-4 strain), cultures were pulsed for 10 min by the addition of [³⁵S]Express label (NEN^TM Life Science Products, Boston, MA) and chased by the addition of excess methionine and cysteine. Cell samples were taken at the end of the pulse period and after 10 and 20 min of chase. Cell lysates were prepared by bead-beat lysis, and labeled species were precipitated from a common extract with specific antibodies for CPY, Gas1p, or anti-HA monoclonal antibody that recognizes Axl2p-HA. For endoglycosidase H (Endo H) experiments, 20-min chase time points were taken, and Axl2p-HA was immunoprecipitated in duplicate from indicated strains. Washed immunoprecipitates were equilibrated with 100 mM sodium citrate, pH 5.5, and one tube of each duplicate was incubated with 5 mU of Endo H (Sigma Chemical Co., St. Louis, MO) for 12 h at 37°C. Samples were resolved on 7.5 or 10% polyacrylamide gels, and labeled species were visualized by fluorography.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Identification of Erv14p
Uncoated ER-derived vesicles were isolated, and vesicle proteins were resolved on a 15% polyacrylamide gel as previously described (Belden and Barlowe, 1996). Approximately 0.02 mg of the 14-kD protein was purified and subjected to automated NH₂-terminal sequencing. The sequence GAWLFILAVVVNXINLFG was obtained from the polypeptide corresponding to Erv14p (X represents a residue that could not be unambiguously determined). The NH₂-terminal sequence of Erv14p (Fig. 1 A, underlined) aligns with an unidentified open reading frame on chromosome VII (YGL054c) reported by the yeast genome sequencing project (Feuermann et al., 1997). Conceptual translation of YGL054c produces a 138–amino acid protein that contains three segments of sufficient length and hydrophobicity to span a lipid bilayer. Based on this sequence information and further data presented in this report, Erv14p is likely an integral membrane protein. A search of the current protein database identified a 142– amino acid unidentified open reading frame in S. cerevisiae, YBR210w (Feldmann et al., 1994), which shares 63% identity with Erv14p, and the predicted 144–amino acid cornichon gene product from Drosophila melanogaster, which shares 36% identity. Other than these sequence homologies, Erv14p does not contain consensus sites for N-linked glycosylation or any characterized sequence motifs to provide insight into molecular function.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 1 (A) Predicted amino acid sequence of Erv14p with underlined amino acids to indicate NH2-terminal residues sequenced. Shown below is the predicted cornichon (cni) polypeptide sequence with amino acid identities and similarities (+) indicated. (B) Hydrophobicity plot as described (Kyte and Doolittle, 1982).

erv14

erv14

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Erv14p is packaged into COPII-coated vesicles. (A) Samples prepared from yeast strains with (lane 1) and without (lane 2) Erv14p-HA (pRS316-ERV14HA) and immunoblotted with anti-HA antibody. Sec61p, a resident ER protein, is shown to indicate equivalent amounts of protein were loaded in lanes 1 and 2. (B) In vitro budding reactions with microsomes prepared from CBY409. One tenth of a total reaction (T) and budded vesicles isolated after incubation with COPII proteins (+) or a mock incubation (–). Samples were resolved on a 12.5% polyacrylamide gel and immunoblotted with antiserums specific for Sec12p (ER resident), Sec22p (v-SNARE), and Erv25p (abundant vesicle protein) as controls or anti-HA antibody to detect Erv14p-HA.

Next, we investigated the nature of Erv14p association with membranes. The predicted amino acid sequence for Erv14p suggests this protein spans the lipid bilayer three times and is consistent with the result that several of the Erv proteins (including an 14-kD species) partition to a carbonate inextractable pellet fraction (Rexach et al., 1994). The fractionation behavior of Erv14p-HA was monitored (Fig. 3) under conditions that extract peripherally bound membrane proteins (2 M urea), release lumenal proteins (0.1 M Na₂CO₃, pH 11), or solubilize integral membrane proteins (1% Triton X-100). The fractionation profile of Erv14p-HA was identical to an integral membrane protein such as Emp47p (Schröder et al., 1995) and not the peripheral membrane protein Sec23p (Hicke and Schekman, 1989). We conclude that Erv14p is an integral membrane protein. Application of the "positive inside" rule (von Heijne and Gavel, 1988) suggests a topology such that the NH₂ terminus is oriented toward the cytoplasm and the COOH terminus is oriented toward the lumenal compartment.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Erv14p is an integral membrane protein. Yeast cell lysates prepared from CBY409 were treated (see Materials and Methods) with buffer, buffer containing 0.5 M NaCl, 2.5 M urea, 0.1 M Na2CO3, pH 11, or 1% Triton X-100, and then centrifuged at 100,000 g. The resulting supernatant fluid (S) or pellet (P) fractions were resolved on 12.5% polyacrylamide gels and immunoblotted for Emp47p (integral membrane protein), Sec13p (peripheral membrane protein), or anti-HA to detect Erv14p-HA.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Erv14p localizes to perinuclear regions. Double-label immunofluorescence microscopy of CBY409 cells using Kar2p antiserum and anti-HA monoclonal antibody for Erv14p-HA detection. Differential interference contrast (DIC) indicates cell boundaries and diamidophenylindole (DAPI) stains nuclei. Note significant overlap in staining pattern of the ER-resident protein Kar2p and Erv14p-HA.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Sucrose gradient fractionation of Erv14p-HA. A whole cell lysate from strain CBY409 was separated on a sucrose density gradient (20–60%) and fractions collected from the top. Relative levels of Sec61p (ER marker), Emp47p (Golgi marker), and Erv14p-HA in each fraction were quantified by densitometry of immunoblots. GDPase (Golgi marker) was measured by enzymatic assay.

ERV14/erv14

erv14

erv14

Erv14p shares amino acid identity with the Drosophila cornichon gene product over the entire open reading frame, and their hydrophobicity profiles are superimposable. The cornichon gene is necessary for establishment of anterior–posterior asymmetry during early stages of oogenesis (Ashburner et al., 1990; Roth et al., 1995); however, a molecular determination of cornichon gene product function has not been reported. We used this clue of cornichon function in generating cellular asymmetry to explore a possible role for Erv14p in yeast cell polarity. S. cerevisiae undergoes polarized cell growth such that the plane of cell division is determined by the site of bud formation and is dependent on cell type. Haploid yeast bud in an axial manner, placing each new bud adjacent to the previous bud site. Genetic analysis of bud site selection has revealed a group of bud and axl mutants defective in the haploid axial budding pattern. The phenotypes of these mutants fall into distinct categories, with one group that displays a random bud site selection pattern and a second group that exhibits bipolar patterns instead of the normal axial pattern (Bender and Pringle, 1989; Chant and Herskowitz, 1991; Chant et al., 1991; Fugita et al., 1994; Chant and Pringle, 1995; Halme et al., 1996; Roemer et al., 1996; Sanders and Herskowitz, 1996). A history of bud site selection in yeast may be visualized by staining with calcofluor, a fluorophore that binds to chitin and reveals previous bud scars as chitin-rich rings (Hayashibe and Katohda, 1973). Logarithmic-stage cultures of wild-type and erv14 strains were grown in rich medium and stained with calcofluor. Strikingly, erv14 haploid strains displayed a nonaxial budding phenotype that was not observed in an isogenic wild-type strain (Fig. 6). In a quantitative analysis of bud site selection, the penetrance of this phenotype was incomplete, but a significant fraction of mothers exhibited nonaxial budding patterns (see Table II). Transformation of erv14 strains with a CEN-based plasmid expressing Erv14p or Erv14p-HA reversed the nonaxial budding phenotype and confirmed that this defect was caused by deletion of Erv14p (Table II). Furthermore, these results indicated ERV14-HA is functional and complements as effectively as unmodified ERV14.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Budding patterns in wild-type (WT) and erv14 strains. Logarithmically grown cells were stained with calcofluor to visualize chitin rings. Wild-type cells display the characteristic axial budding pattern, whereas erv14 cells exhibit nonaxial budding patterns.

erv14

erv15

erv14

Wild-type a/ diploids exhibit a bipolar budding pattern such that a mother cell may produce a new bud at either pole from the previous bud site. We examined the budding pattern in homozygous diploid strains lacking Erv14p (CBY410) and in strains lacking both Erv14p and Erv15p (CBY411). These strains did not exhibit any alterations in the bipolar positioning of the bud site (data not shown). Therefore, erv14 strains possess haploid-specific defects in establishing cell polarity, as reported for a subset of the axl and bud mutants (Chant et al., 1995; Roemer et al., 1996).

Axl2p Accumulates in the ER of erv14 Mutants
Because Erv14p is localized to the early compartments of the secretory pathway, and this pathway is responsible for the delivery of proteins to the plasma membrane, we considered the possibility that Erv14p was required for transport of factors to the cell surface for establishment of cell polarity. Combined molecular/genetic approaches have provided a wealth of information on the proteins involved in bud site selection in yeast (for review see Chant, 1996). Axl2p is one of the characterized proteins that is required for an axial budding pattern and displays a bipolar phenotype when disrupted in haploid cells. Axl2p is also an integral membrane glycoprotein with a predicted type I topology and is known to traverse the secretory pathway en route to the cell surface (Roemer et al., 1996). During the biogenesis of Axl2p, N-linked core oligosaccharides are added in the ER and are further extended during passage through Golgi compartments. We tested if Axl2p was efficiently transported in strains carrying an erv14 allele and used a triple-HA–tagged version of Axl2p (known to complement an axl2 strain) to monitor transport of Axl2p (Roemer et al., 1996). In an initial experiment, we immunoblotted whole-cell extracts from wild-type and mutant strains expressing Axl2p-3HA (Fig. 7). In an erv14 null strain, an 150-kD HA-tagged species that was not detected in wild-type strains was apparent. In contrast, wild-type strains expressed an 220-kD form that was a very minor species in erv14 null strains. We reasoned that the 150-kD form of Axl2p-3HA that was unique to the erv14 strain represented a core-glycosylated form of this secretory protein that may accumulate in the ER. The next series of experiments are focused on this hypothesis.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Immunoblot of Axl2p-HA in wild-type and erv14 strains. Lysates prepared from wild-type strain CBY461 (lane 1), erv14 strain CBY463 (lane 2), and sec12 strain CBY471 (lanes 3 and 4) were resolved on a 7.5% polyacrylamide gel. For the sec12 strain, cells were grown at 25°C (lane 3) or shifted to 37°C for 2 h (lane 4). Proteins were transferred to nitrocellulose and probed with anti-HA antibodies for Axl2p-HA detection. Note the wild-type form of Axl2p-HA (*) and a faster migrating form of Axl2p-HA (**) observed in the erv14 strain (lane 2) and the sec12 strain shifted to a restrictive temperature (lane 4). A faint cross-reactive species migrating just below the faster migrating form of Axl2p-HA is an endogenous protein detected in strains lacking AXL2::3XHA.

The above experiments suggest that Axl2p-3HA accumulates in the ER as a 150-kD core-glycosylated form in erv14 and sec12-4 strains. The immunoblot experiments provide a steady-state view of Axl2p-3HA in these strains. However, we sought to characterize the kinetics of Axl2p-3HA transport in wild-type and erv14 strains through a pulse-chase analysis. In these experiments, cells were grown in minimal medium and pulsed with [³⁵S]methionine and cysteine for 10 min to label newly synthesized proteins. Excess cold methionine and cysteine were then added to initiate the chase phase, and the fate of specific secretory proteins (Axl2p-3HA, CPY, and Gas1p) were monitored by selective immunoprecipitation of these species from whole-cell lysates (Fig. 8). CPY is first detected in the ER as the P1 precursor (67 kD), then modified upon arrival to the Golgi complex producing the P2 form (69 kD), and finally processed to the mature form (67 kD) in the vacuole (Stevens et al., 1984). As shown in Fig. 8, wild-type strains and the erv14 strain exhibited similar kinetics for CPY transport to the vacuole, whereas the sec12-4 temperature-sensitive mutation accumulated the ER form (P1) of CPY when shifted to a restrictive temperature. Similar results were observed for transport of the glycosylphosphatidylinositol-anchored plasma membrane protein Gas1p. Newly synthesized Gas1p appeared in the ER as a 105-kD glycosylphosphatidylinositol-anchored precursor that carries N- and O-linked oligosaccharide. As Gas1p traverses the Golgi complex, outer chain glycosylation residues are added, generating the 125-kD mature form (Nuoffer et al., 1991). Gas1p was transported out of the ER in wild-type and erv14 strains; however, we see a subtle delay in Gas1p transport (note the ratio of ER form to mature form after a 10-min pulse in the Gas1p panel in Fig. 8). This delay was not as severe as seen in strains bearing deletions of other Erv proteins, such as Emp24p and Erv25p (Belden and Barlowe, 1996). In sec12-4 cells, the ER form of Gas1p accumulated but displayed a reduced electrophoretic mobility during the chase period, as has been previously observed (Schimmöller et al., 1995; Belden and Barlowe, 1996). Based on these results, we conclude that the ERV14 gene is not required for transport of CPY and Gas1p from the ER to the Golgi complex, in contrast to sec mutant strains such as sec12-4 (Novick et al., 1980; Stevens et al., 1984). This result is also consistent with the wild-type growth rate for erv14 since cell surface expansion (i.e., growth rate) was not impeded in these strains, again in contrast to typical sec mutations that are lethal and prevent expansion of the plasma membrane.

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Pulse-chase analysis of secretory proteins in WT and erv14 strains. sec12 strain CBY471, wild-type strain CBY461, and erv14 strain CBY463 were pulsed for 10 min followed by a chase of 10 and 20 min. Antibodies specific for Axl2p-HA, CPY, and Gas1p were used to immunoprecipitate labeled protein from a common extract. Immunoprecipitates were washed and resolved on 7.5 or 10% polyacrylamide gels, and labeled proteins were visualized by fluorography.

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We proposed that Axl2p is not exported from the ER in erv14 strains and sought additional lines of evidence in support of this proposal. Axl2p acquires N-linked oligosaccharide in the ER of yeast cells (Roemer et al., 1996), and if the form that accumulates in an erv14 strain is trapped in the ER, treatment with Endo H should liberate this covalently linked carbohydrate and produce a faster migrating polypeptide on polyacrylamide gels (Orlean et al., 1991). After immunoprecipitation of Axl2p-3HA from various strains, treatment with Endo H produced an alteration in the mobility of the labeled polypeptide (Fig. 9). The forms of Axl2p-3HA that accumulated in an erv14 and a sec12-4 strain displayed identical properties upon treatment with Endo H, shifting the 150-kD ER-form to an 125-kD species. Based on an approximation of each core oligosaccharide contributing 2 kD in mass (Orlean et al., 1991), we estimate the attachment of 13 N-linked chains on Axl2p, which was predicted to contain 16 potential N-linked glycosylation sites (Roemer et al., 1996). Treatment of the mature form of Axl2p-3HA with Endo H also produced a distinct shift in size from 220 to 145 kD, but clearly not to the 125-kD species that appears after removal of N-linked residues from the ER form. We speculate that Axl2p acquires O-linked oligosaccharides in the ER that are then extended upon transport through the Golgi (Tanner and Lehle, 1987). Indeed, preliminary data indicate that Axl2p contains O-linked oligosaccharide (Sanders, S., and M. Gentzsch, personal communication), which would also explain the difference between the predicted size (90 kD) and the Endo H–treated ER form (125 kD). Regardless, the form of Axl2p-3HA that accumulates in an erv14 strain behaves identically to the ER form that accumulates in a sec12-4 strain, a well-characterized mutant representative of mutants blocked in export from the ER (Novick et al., 1980; Stevens et al., 1984; Nakano et al., 1988). This observation, coupled with the fact that CPY and Gas1p are transported and modified correctly, make it unlikely that the erv14 mutation alters the glycosylation machinery producing modification defects on Axl2p.

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Figure 9 Endo H treatment of Axl2p. Immunoprecipitates of Axl2p-HA from sec12 (CBY471), wild-type (CBY461), and erv14 (CBY463) were incubated in the absence (–) or presence (+) of Endo-H. Samples were resolved on a 7.5% polyacrylamide gel, and labeled species were visualized by fluorography.

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View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Figure 10 Axl2p is mislocalized in erv14 strains. Double-label immunofluorescence microscopy of wild-type (CBY461) and erv14 (CBY463) strains with Kar2p antiserum and anti-HA monoclonal antibody for Axl2p-HA detection. Note the distinct staining pattern for Axl2p-HA observed in wild-type cells that is absent in erv14 strains.

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View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 11 Sucrose gradient fractionation of Axl2p-3HA. Whole-cell lysates from wild-type strain CBY461 (A and B) and erv14 strain CBY463 (C and D) were separated on sucrose density gradients (20– 60%) containing magnesium, and fractions were collected from the top. Relative levels of Sec61p (ER marker), PMA, Vph1 (vacuolar marker), and Axl2-3HA in each fraction were quantified by densitometry of immunoblots. GDPase (Golgi marker) was measured by enzymatic assay.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 12 EDTA/sucrose gradient fractionation of Axl2p-3HA. Whole-cell lysates from wild-type strain CBY461 (A and B) and erv14 strain CBY463 (C and D) were separated on sucrose density gradients (20–60%) containing 10 mM EDTA, and fractions were collected from the top. Relative levels of Sec61p (ER marker), PMA, Vph1 (vacuolar marker), and Axl2-3HA in each fraction were quantified by densitometry of immunoblots. GDPase (Golgi marker) was measured by enzymatic assay.

Pleiotropic Effects of erv14 Mutations

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	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Organelles of the eukaryotic secretory pathway remain distinct in spite of dynamic anterograde and retrograde transport processes. Specific coat proteins that catalyze transport between the ER and Golgi are thought to contribute to this organization (Schekman and Orci, 1996), although the molecular mechanisms that link coat proteins with cellular sorting are not understood. Here we characterize a novel protein, termed Erv14p, that was discovered on ER-derived transport vesicles and appears to participate in sorting during ER to Golgi transport. Erv14p is an integral membrane protein that shares significant sequence identity (36%) and similarity (63%) over the entire length of the Drosophila cornichon gene product. Localization studies indicate that Erv14p resides in the ER and Golgi compartments of cells and is selectively packaged into ER-derived vesicles in a COPII-dependent manner. Disruption of the ERV14 gene produces viable haploid cells that exhibit defects in axial bud site selection. This phenotype correlates with the failure of erv14 strains to transport a bud site selection protein (Axl2p) to the cell surface. We propose that Erv14p regulates export of Axl2p and possibly other secretory proteins from the ER in COPII-coated vesicles as depicted in Fig. 13.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 13 Model for Erv14p function in intracellular transport. In a wild-type strain (WT), Axl2p is efficiently packaged into ER-derived vesicles, and axial bud sites are established. In an erv14 strain, ER-derived vesicles are produced, but Axl2p fails to enter vesicles, and cells bud in a nonaxial manner.

Surprisingly, deletion of ERV14 did not interfere with bulk secretory function but instead produced defects in yeast cell polarity. To comprehend this phenotype, we reviewed the literature concerning bud site selection in yeast. Haploid yeast cells exhibit an axial budding pattern whereby each new bud forms directly adjacent to the previous bud site. The selection of an appropriate bud site is thought to rely on a series of integrated events: first, the selected site for budding must be marked; second, components required for bud formation are assembled at this site; and third, the actin cytoskeleton and secretory apparatus are directed toward the selected site for growth of the emerging bud (Drubin, 1991). For the axial budding pattern, it has been proposed that this first stage is accomplished through a landmark that persists after cytokinesis and provides the cell with an assembly site for the next round of budding (Chant and Herskowitz, 1991; Snyder et al., 1991). Axl2p is unique among the characterized bud site selection proteins because it must be delivered to the plasma membrane via the secretory pathway. Once anchored in the plasma membrane, Axl2p may recruit additional components to the incipient bud site. In haploid axl2 strains, the axial budding pattern is lost, and a bipolar phenotype is observed such that 50% of the newly formed buds are placed at the opposite pole from the previous bud site (Halme et al., 1996; Roemer et al., 1996).

When we examined erv14 strains, we observed a nonaxial budding pattern and found that Axl2p accumulated in the ER. The accumulation of Axl2p in the ER provides a rational explanation for the nonaxial phenotype displayed by erv14 strains. Based on the following observations, however, we suspect that some Axl2p is slowly transported to the cell surface in erv14 strains. First, immunoblot analysis indicated that a low level of the mature form of Axl2p was detected in erv14 strains, although this species appears to be synthesized at a slow rate since it was not detected in pulse-chase experiments. Second, only 16% of the erv14 cells exhibit budding from both poles in contrast to a more typical bipolar pattern observed in axl2 strains (Halme et al., 1996; Roemer et al., 1996). This may be explained if erv14 cells deliver some Axl2p to incipient bud sites, but with suboptimal levels of plasma membrane Axl2p, a nonaxial phenotype is displayed intermittently. Third, we find that overexpression of Axl2p partially suppressed the nonaxial budding phenotype in erv14 strains (Table II). Again, this observation may be explained if some Axl2p trickles through the secretory pathway in an erv14 strain, and overexpression of Axl2p increases the level of this flow.

Could the role of Erv14p in yeast trafficking provide insights on the function of cornichon? The cornichon mutants display phenotypes that are similar to torpedo (EGF receptor) and gurken (TGF homologue and putative torpedo ligand) mutants. These three genes are components of an intercellular signaling process between germ-line cells and surrounding follicle cells that establishes anterior–posterior polarity during early stages of oogenesis. Both gurken and cornichon are required in germ-line cells, whereas torpedo function is required in surrounding follicle cells of the developing egg chamber. The localized delivery of gurken protein to the plasma membrane of the oocyte presumably activates the EGF receptor torpedo in a subset of follicle cells and specifies their posterior fate. In turn, the polarized arrangement of the surrounding follicle cells provides spatial information to the oocyte and is critical for the reorganization of the microtubule network at mid-oogenesis. In cornichon, gurken, and torpedo mutants, intercellular signaling between the oocyte and surrounding follicle cells fails. Anterior markers are abnormally expressed in the posterior follicle cells, producing egg chambers that have the normal arrangement of oocyte and nurse cells but have duplicated anterior follicle cells at both poles. It is postulated that the gurken signal emanating from the oocyte represses anterior fates in adjacent follicle cells and directs them to a posterior fate (Gonzáles-Reyes and St. Johnston, 1994; Roth et al., 1995). In cornichon mutants, gurken mRNA expression and perinuclear localization is normal. Examination of gurken protein reveals that the expression level is normal, but cornichon egg chambers display a diffuse staining pattern for gurken protein at the oocyte membrane, unlike the tight stripe-like distribution at the dorsal–anterior corner of the oocyte observed in wild-type. Indeed, one explanation offered for this phenotype was that an altered polarity in cornichon egg chambers would cause less efficient membrane targeting of vesicles transporting gurken protein (Roth et al., 1995).

In light of our findings with erv14 strains, it is plausible that in cornichon mutants, a landmark on the oocyte cell surface is not established to correctly orient the secretory pathway. Much as the yeast cell uses Erv14p to export Axl2p from the ER and mark the axial bud site, we speculate that cornichon is required for export of a polarity establishment factor from the oocyte ER in Drosophila. Although there are no candidates for this factor at present, the high degree of homology shared between Erv14p and the cornichon gene product suggests this molecule may be related to Axl2p.

The mechanism by which Erv14p catalyzes Axl2p export from the ER remains to be determined, and we can envision several possibilities to explain this transport block. First, Erv14p could directly bind to Axl2p and escort this protein out of the ER, acting as an adaptor for incorporation of Axl2p into COPII-coated vesicles. Direct binding could also be involved in the folding of Axl2p or assembly of Axl2p into an oligomeric complex before exit from the ER, although we do not favor this possibility because proteins involved in these processes (e.g., BiP) appear to remain in the ER and are not selectively packaged into ER-derived vesicles (Salama et al., 1993; Barlowe et al., 1994; Rexach et al., 1994). Furthermore, the forms of Axl2p that accumulate in the ER of erv14 and sec12 strains are indistinguishable and are not rapidly degraded, suggesting that Axl2p is stably folded but unable to depart the ER. The erv14 phenotype resembles yeast strains that lack Shr3p, a resident ER protein that is required for the export of amino acid permease molecules in COPII-coated vesicles (Ljungdahl et al., 1992; Kuehn et al., 1996, 1998). However, the activities of Erv14p and Shr3p appear to be distinct because Erv14p is selectively packaged into COPII-coated vesicles, whereas Shr3p does not appear to enter these vesicles (Kuehn et al., 1996). Another set of ER proteins that produce selective transport defects (Vma12p, Vma21p, and Vma22p) are required for export of integral membrane subunits of the vacuolar ATPase (Hill and Stevens, 1994, 1995; Jackson and Stevens, 1997). It remains to be determined if these Vma proteins are selected for incorporation into COPII-coated vesicles. However, it has been proposed that Vma12p, Vma21p, and Vma22p function in the assembly of V-ATPase subunits in the ER and that assembly is a prerequisite for export from the ER (Jackson and Stevens, 1997). At present, we are aware of only three proteins that when deleted produce selective ER to Golgi transport defects and are enriched on COPII-coated vesicles: Emp24p (Schimöller et al., 1995), Erv25p (Belden and Barlowe, 1996), and Erv14p. We continue to explore the protein–protein interactions and mechanisms by which these molecules influence sorting during transport from the ER.

	Acknowledgments

 
We thank Bill Belden for help with strain construction, Terry Roemer and Michael Snyder for generously providing AXL2 constructs, Hannele Ruohola and Trudi Schüpbach for helpful discussions on Drosophila development, and Sylvia Sanders for advice and sharing her unpublished observations.

This work was supported by grants from the National Institute of General Medical Sciences and the Pew Scholars Program in the Biomedical Sciences.

Submitted: 29 April 1998

Revised: 9 July 1998

Address all correspondence to Charles Barlowe, Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755. Tel.: (603) 650-6516. Fax: (603) 650-1353. E-mail: barlowe{at}dartmouth.edu

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