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Vol. 17, Issue 12, 4988-5003, December 2006

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The Yeast Tumor Suppressor Homologue Sro7p Is Required for Targeting of the Sodium Pumping ATPase to the Cell Surface

Ingrid Wadskog^*,, Annabelle Forsmark^*, Guendalina Rossi, Catherine Konopka, Mattias Öyen^||, Mattias Goksör^¶, Hans Ronne^||,#, Patrick Brennwald, and Lennart Adler^*

*Department of Cell and Molecular Biology, Microbiology, Göteborg University, SE-405 30 Göteborg, Sweden; Department of Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, NC 27599; Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706; ^||Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden; ^¶Department of Physics, Göteborg University, SE-412 96 Göteborg, Sweden; and ^#Department of Medical Biochemistry and Microbiology, Uppsala University, SE-751 23 Uppsala, Sweden

Submitted August 24, 2005; Revised August 25, 2006; Accepted September 18, 2006
Monitoring Editor: Akihiko Nakano

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The SRO7/SOP1 encoded tumor suppressor homologue of Saccharomyces cerevisiae is required for maintenance of ion homeostasis in cells exposed to NaCl stress. Here we show that the NaCl sensitivity of the sro7 mutant is due to defective sorting of Ena1p, the main sodium pump in yeast. On exposure of sro7 mutants to NaCl stress, Ena1p fails to be targeted to the cell surface, but is instead routed to the vacuole for degradation via the multivesicular endosome pathway. SRO7-deficient mutants accumulate post-Golgi vesicles at high salinity, in agreement with a previously described role for Sro7p in late exocytosis. However, Ena1p is not sorted into these post-Golgi vesicles, in contrast to what is observed for the vesicles that accumulate when exocytosis is blocked in sec6-4 mutants at high salinity. These observations imply that Sro7p has a previously unrecognized role for sorting of specific proteins into the exocytic pathway. Screening for multicopy suppressors identified RSN1, encoding a transmembrane protein of unknown function. Overexpression of RSN1 restores NaCl tolerance of sro7 mutants by retargeting Ena1p to the plasma membrane. We propose a model in which blocked exocytic sorting in sro7 mutants, gives rise to quality control-mediated routing of Ena1p to the vacuole.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Targeted delivery of secretory vesicles to discrete regions of the plasma membrane is essential for establishment and maintenance of cell polarity (Nelson and Yeaman, 2001; Pruyne et al., 2004). The tumor suppressor lethal(2)giant larvae (Lgl) has an important role in establishing cell polarity in developing Drosophila embryos (Wodarz, 2000; Humbert et al., 2003; Bilder, 2004). Homozygous inactivation of the lgl gene results in improper targeting of selected epithelial transmembrane proteins to their correct apical destination (Bilder et al., 2000; Hutterer et al., 2004; Wirtz-Peitz and Knoblich, 2006), leading to defects in apical-basal polarity. Lgl also plays an important role in asymmetric cell division by contributing to the polarization of cytosolic fate determinants in mitotic neuroblasts (Ohshiro et al., 2000; Peng et al., 2000; Betschinger and Knoblich, 2004). Lgl deficiency therefore results in improper cell fate specification and failure to differentiate correctly. The loss of cell polarity in lgl mutants is proposed to be a primary reason for disruption of larval tissue organization and for the massive overgrowth of the larval brain and the imaginal discs (Bilder, 2004). Hence, Lgl appears to be involved in a pathway that coordinates protein sorting, cell polarity, and growth control.

The Lgl protein is the founding member of a growing family of WD-40 repeat proteins conserved in eukaryotes ranging from yeast to man (Tomutsune et al., 1993; Strand et al., 1995; Koyama et al., 1996; Kagami et al., 1997; Larsson et al., 1998; Grifoni et al., 2004). Studies in yeast have provided clues to the molecular function of these proteins, implicating involvement in late stages of polarized secretion (Kagami et al., 1998; Lehman et al., 1999; Gangar et al., 2005; Zhang et al., 2005). Current models state that a conserved multisubunit complex, known as the exocyst, assembles as cargo vesicles arrive at the exocytic sites, establishing the primary connection between the plasma membrane and the secretory vesicle (TerBush et al., 1996; Guo et al., 2000; Wiederkehr et al., 2004). After this vesicle tethering, the coiled-coil SNARE (soluble N-ethylmalemide–sensitive factor attachment protein receptor) proteins, opposed on vesicle (v-SNAREs) and target membranes (t-SNAREs), form a complex that mediates a short-range interaction between the vesicle and plasma membranes. Studies in yeast have revealed that the two yeast Lgl homologues Sro7p and Sro77p (also named Sop1p and Sop2p) interact with myosins (Kagami et al., 1998, Gangar et al., 2005) components of the exocyst (Lehman et al., 1999; Zhang et al., 2005; Grosshans et al., 2006), and Sec9p (Lehman et al., 1999; Gangar et al., 2005), a t-SNARE protein involved in docking of post-Golgi transport vesicles with the plasma membrane. Deletion of both SRO7 and SRO77 leads to a cold-sensitive phenotype (Kagami et al., 1998) that is characterized by defective secretion and intracellular accumulation of post-Golgi vesicles (Lehman et al., 1999).

We isolated the yeast SOP1/SRO7 gene (hereafter referred to as SRO7) by complementation of a salt-sensitive Saccharomyces cerevisiae mutant (Larsson et al., 1998; Wadskog et al., 2004). Mutants having a deleted SRO7 gene are specifically sensitive to NaCl stress and exhibit increased intracellular levels of Na⁺ when exposed to high NaCl media. The sro7 sro77 double mutant is hypersensitive to NaCl, and this phenotype is partially rescued by ectopic expression of the Drosophila lgl gene, indicating functional conservation of the homologues (Larsson et al., 1998). The primary pathway for sodium export in S. cerevisiae occurs via the plasma membrane P-type ATPase encoded by the ENA1/PMR2 gene (Garciadeblas et al., 1993; Wieland et al., 1995). Expression of ENA1 is vital for maintenance of intracellular ion homeostasis in salt-stressed yeast cells and is subject to a complex network of regulatory interactions involving not only salt or pH stress but also general metabolic processes (Serrano et al., 1999; Wadskog and Adler, 2002). A second Na⁺ extrusion system in S. cerevisiae is the cation/proton antiporter encoded by NHA1 gene (Prior et al., 1996; Banuelos et al., 1998). This transporter becomes important for Na⁺ efflux only at acidic external pH values, whereas its expression does not seem to be controlled by salt stress or changes in pH.

In this article, we demonstrate that the salt sensitivity of sro7 mutants is due to failure of the ENA1-encoded sodium pump to be expressed on the cell surface. Instead, Ena1p is diverted for vacuolar degradation in a manner dependent on ubiquitin ligase and the vacuolar protease Pep4p. The observed mis-targeting of Ena1p in salt-stressed mutants is accompanied by accumulation of secretory vesicles. However, defective sorting only appears to involve a fraction of the vesicle-cargo-proteins. A number of plasma membrane proteins examined are correctly sorted in salt-stressed sro7 mutants. We have also identified a novel gene, RSN1, which functions as a multicopy suppressor of the salt-sensitive growth of sro7 mutants by restoring targeting of Ena1p to the cell surface. We postulate that Sro7p is involved in the molecular machinery required for targeting of Ena1p to the plasma membrane and that Ena1p is identified by the Golgi quality control system and rerouted to the vacuole for degradation in the absence of Sro7p.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Strains, Plasmids, and Growth Conditions
All strains and plasmids used in this study are listed in Tables 1 and 2. The wild-type strain used in most experiments was W303-1A and the congenic sro7 mutant was WKL-2A (Table 1). Unless otherwise stated, strains were grown at 30°C in rich YP media with 20 g/l of glucose, pH 6.5. Defined CBS medium was composed as previously described (Verduyn et al., 1992). For salt induction, appropriate volumes of medium containing 2 M NaCl were added to the cultures to obtain the final NaCl concentration.

Northern Blot Analysis
For Northern analysis of ENA1 mRNA, cells were grown to midlog phase in basal medium before being subjected to NaCl stress. Cells were harvested on ice at time points indicated, and total RNA was isolated by standard procedures. The amount of RNA was determined by spectrophotometry and semiquantitatively using ethidium bromide stained agarose gels. Samples containing 15 µg of total RNA were denatured and run on low formaldehyde (2.5% vol/vol) agarose gels. Blotting onto positively charged nylon membranes was preformed by capillary transfer, using 10x SSC as transfer buffer. The filters were prehybridized for 4 h at 60°C in 5x SSC, 10 mM sodium phosphate (pH 6.5), 10x Denhardt's solution, 2% SDS, and 100 µg/ml herring sperm DNA. Hybridization was preformed at 60°C for 8–10 h in the same solution supplemented with 10% PEG4000. Probes were labeled and added at 5 ng/ml. For detection of the ENA1 transcript a 5'-AAT CAT GCA TGG CAA ACG AGA TTA CT-3' probe, which shows little identity to other ENA genes, was used. As a loading control we used IPP1 mRNA detected using a 5'-TGT CTG GTA GTG TAG GTC ATT AGT-3' probe. Signal intensities were quantified with a Phosphoimager (Molecular Dynamics, Sunnyvale, CA; 425E), using the manufacturer-supplied program, Image Quant.

Tagging of Ena1p
For epitope tagging of the genomic copy of ENA1, a three times–repeated hemagglutinin (HA) epitope was integrated in the ENA1 gene using PCR amplification of a tag-URA3-tag cassette in the pMPY-HA vector, kindly provided by Dr. B. Futcher (CSHL; Schneider et al., 1995). The PCR primers were designed such that 16 bases were complementary to pBluescript sequences of pMPY-HA, whereas flanking regions of 50 bases were complementary to and in frame with the 3'-end of the ENA1 gene. The PCR reaction was carried out using 50 ng of linearized pMPY-HA, 15 pmol of each primer, 0.5 mM dNTPs, 5 µl 10x PCR buffer 1 (Long template PCR kit, Roche, Indianapolis, IN) and 0.5 µl DNA polymerase mix (Long template PCR kit, Roche) in a total volume of 50 µl. The PCR reaction conditions were as follows: 5 min at 94°C, 1 min at 50°C, 3 min at 72°C for one cycle; 1 min at 94°C, 1 min at 50°C, and 3 min at 72°C for nine cycles. PCR products were purified by QIAquick PCR Purification kit, (QIAGEN, Chatsworth, CA), and 1 µg of purified DNA was used for transformation. After selection for stable transformants on medium lacking uracil, positive clones were cultured in YPD overnight to allow for URA3 gene loop out by recombination between the repeated epitope regions. Cells from this culture were transferred to 5-fluoro-orotic acid plates (5-FOA, 1.0 g/l), and resistant colonies were examined by PCR and Western analysis to confirm correct tagging of ENA1. The CEN plasmid pUG35-ENA1 containing ENA1-GFP behind the methionine repressible MET25 promoter was a generous gift from Drs. M. Cyert and Victoria Heath.

Quantitative PCR
To determine SRO77 expression 0.5 µg of total RNA was used for reverse transcription using Bio-Rad iScript First Strand Synthesis kit according to the manufacturers instructions (Richmond, CA). The reaction was scaled down to 10 µl, and each sample was reverse transcribed in duplicate. A pool of samples was used as control for genomic DNA contamination. For the detection of SRO77, the forward primer 5'-GGGCAAAAGCAAATAGAGGT-3', and reverse primer 5'-CAATCAGCATCCAATCCAAG-3', were used. For the detection of the control IPP1, the forward primer 5'-CGTAAGCCACCCAGAAACTAA-3' and reverse primer 5'-ATCGGTCTCACCTTCATCCA-3', were used. Quantitative PCR was performed on the Stratagene Mx3005p, using BD QTaq Polymerase mix with 0.4 µM of each primer and 0.25x SYBR Green I (Molecular Probes, Eugene, OR). cDNA corresponding to 25 ng of RNA was used in each PCR reaction. Denaturation for 2 min at 95°C was followed by 45 cycles of 20s at 95°C, 20s at 60°C, and 20s at 72°C. The fluorescence detection was performed at the 72°C step. After amplification dissociation curve analysis was performed to verify the product formed. Statistic analysis was performed using Excel 2003 (Microsoft, Redmond, WA). The IPP1 gene was used as a reference to calculate the relative expression of SRO77.

Western Blot Analysis
For immunoblot analysis cells cultured to midlog phase in YPD medium were subjected to a step increase in NaCl concentration, and cell samples harvested by centrifugation were frozen in liquid nitrogen. To lyse the cells, pellets were resuspended in 2 M NaOH (300 µl/OD₆₁₀) and incubated on ice for 10 min. The cell suspensions were then neutralized by the addition of trichloroacetic acid to a final concentration of 40%. Before a subsequent 10 min incubation on ice, cells were centrifuged and washed twice in 1 M Tris-HCl, pH 8.0 and resuspended in 2x Laemmli buffer (100 mM Tris-HCl, pH 6.8, 8% SDS, 40% glycerine, 2% mercaptoethanol, 0.005% bromophenol blue). Samples corresponding to 10 ml of OD₆₁₀ = 0.5 were incubated at 50°C for 5 min, loaded onto 10% polyacrylamide criterion gels (Bio-Rad), resolved by SDS-PAGE, and blotted onto nitrocellulose membranes. After transfer, the filters were stained for 5 min in Ponceau S and washed with distilled H₂O to observe the quality of the transfer and check the total amount of protein. HA-tagged Ena1p was detected using anti-HA antibodies (Roche) at 1:2000 dilution as the primary antibody and anti-mouse HRP-conjugated antibody (Roche) at 1:4000 dilution as the secondary antibody. Blots were developed using Lumi-light chemiluminescence detection system (Roche). Immunoblot analysis of Bgl2p (Adamo et al., 1999) and Sso1/2p and Snc1/2p (Lehman et al., 1999) was performed as previously described.

Tagging of Rsn1p, Gap1p, and Suc2p with Fluorescent Tracers
Genomic tagging of RSN1 with the cyan fluorescent protein (CFP) was conducted according to the procedure described by the Yeast Resource Center, University of Washington (http://depts.washington.edu/yeastrc). The integration cassette from plasmid pDH3 was amplified by PCR with the following primers: 5'-GCG GAC CCA AAG TAT AAG GAA GAA GAG AGT CGT TCG GCA GTG GTC GAC GGA TCC CCG GG-3' and 5'-AAA CAA ATA GGA ATA GAT AAT TTC TGA TAA TGT TTA ACC TTT AGA TCG ATG AAT TCG AGC-3', which contain sequences from the 3' end of the RSN1 gene (underlined) and sequences from the pDH3 plasmid. Wild-type cells were transformed with the PCR product and plated on selective YPD G418 medium. Positive clones were further verified by PCR. Finally the region of integration was sequenced to ascertain that the proper reading frame was used.

To tag Gap1p by green fluorescent protein (GFP) on its C-terminus, PCR was used to synthesize a fragment containing the GAP1 coding sequence lacking its stop codon. The primers used were 5'-AAA AAA CCC GGG ATG AGT AAT ACT TCT TCG TA and CGG AGT ATC GAT ACA CCA GAA ATT CCA GAT TC-3', containing SmaI and ClaI restriction sites, respectively. After digestion, the fragment was cloned into the plasmid pUG35 (URA3 CEN plus gene encoding the yeast-enhanced GFP), which was cleaved with corresponding enzymes, to produce the GAP1-GFP fusion. The construct was confirmed by restriction enzyme analysis and fluorescence microscopy.

Tagging of Suc2p by GFP on its C-terminus was performed by the same procedure using the primers 5'-AAA AAA ACT AGT CTC TCA GAG AAA CAA GCA-3' and 5'-AAA AAA GAA TTC CTT CCC TTA CTT GGA AC-3' to generate a SUC2 fragment flanked by Spe1 and EcoR1 restriction sites. The fragment was cloned into the pUG35 plasmid as described for pUG35-GAP1.

Fluorescence Microscopy
Cells taken from exponential growth phase in defined media were subjected to conditions as described in Results and viewed with the Leica DMRXA microscope (Deerfield, IL) using a Fluotar lens and the Leica 513852 filter for GFP or CFP fluorescence. To obtain a representative picture of the appearance of the average cell in the culture >1000 cells were observed for each condition shown. Photographs were captured using a Hamamatsu ORCA cooled CCD camera (Bridgewater, NJ). For N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64, Molecular Probes) labeling of yeast vacuoles, exponentially growing cells were incubated at 30°C with 32 µM FM4-64 for 15 min, followed by washing and resuspension in fresh medium for 1–1.5 h (Vida and Emr, 1995), before expression of ENA1 was induced.

Confocal Microscopy
The CFP-tagged cells were imaged using a laser scanning confocal unit (MRC1024, Bio-Rad) mounted on the side port of an inverted microscope (TE-300, Nikon, Melville, NY). The CFP was excited using a fiber-coupled blue diode laser (405 nm). The samples were imaged using a 100x Plan Fluor objective (Nikon) with a numerical aperture of 1.3. A longitudinal resolution of 1.2 µm was obtained by means of the adjustable confocal pinhole. Because of the weak CFP signal a 405-nm Raman Notch Filter (FWHM 10 nm; Melles Griot, Rochester, NY) was used to prevent the excitation light of reaching the detector. Thus, no additional emission filters was used. The signal was collected by using a five-frame Kalman collection filter. Relative fluorescence was measured using LaserPix software (Bio-Rad).

Electron Microscopy
Wild-type and sro7 strains were grown to midlog phase in YPD at 25°C. Cells were shifted for 2 h in YPD or YPD/0.6 M NaCl media and processed for electron microscopy (EM) as described previously (Adamo et al., 1999).

Secretion Assays
Bgl2p secretion assays were performed as described previously (Adamo et al., 1999). The examined strains were grown to midlog phase in YPD at 25°C and then shifted to YPD or YPD + 0.6 M NaCl media for 2 h. Invertase secretion assays were performed with cells transformed with the pUG35-SUC2 plasmid. Cells were grown to midlog phase in CBS medium at 25°C and then incubated for 2 h in methionine-free CBS or CBS + 0.6 M NaCl media to de-repress SUC2 expression. Invertase secretion was assayed as previously described (Lehman et al., 1999).

Suppressor Screen
To isolate multicopy suppressor genes of sro7 salt sensitivity, the mutant strain was transformed with the pHR81-based yeast genomic library (Nehlin et al., 1989). Transformants were first selected on YNB plates lacking uracil and then replica plated onto YPD containing 0.7 M NaCl. Positive clones, which contained plasmid inserts that restored growth of the sro7 mutant at high salinity, were isolated. After plasmid rescue and retransformation, library inserts were partially sequenced and compared against the yeast genome database via BLAST searches. For those inserts containing multiple open reading frames (ORFs), each ORF was subcloned into pRS426 and transformed into sro7. A total of 12,000 transformants were screened.

Subcellular Fractionation
To localize Rsn1p by fractionation, wild-type W303-1B cells harboring CFP tagged Rsn1p were grown to OD₆₁₀ 0.5–1.0 in 200 ml YPD medium. Cells were washed by centrifugation and resuspended in twice the pellet volume of resuspension buffer (R-buffer), containing 50 mM Tris, pH 7.5, 5 mM EDTA, Complete protease inhibitor cocktail (Boehringer Mannheim, Germany, 1 tablet per 50 ml cell extract), and 0.1 M KCl. Four volumes of glass beads (0.5 mm ) were added, and the cells were then processed in a bead beater four times for 30 s. Unlyzed cells were removed by centrifugation at 500 x g for 5 min at 4°C. The lysate was divided into three aliquots and diluted with an equal volume of either R-buffer, R-buffer with 2.0 M KCl, or R-buffer with 2% Triton X-100. Centrifugation at 10,000 x g for 10 min resulted in pellets (P10) and supernatants (S10). P10 was resuspended in R-buffer, and S10 was further centrifuged at 100,000 x g for 1 h in a Beckman TL-100 ultracentrifuge (Berkeley, CA). The resulting pellets were resuspended in R-buffer. Aliquots containing equal amounts of cell material were boiled for 1 min in 2x Laemmli buffer (see above) and loaded to 10% Tris-HCl criterion gels (Bio-Rad). Immunoblot analysis was performed as previously described for Ena1p-HA using anti-GFP as the primary antibody (kindly provided by Dr. Pam Silver).

Post-Golgi vesicles were isolated as previously described (Brennwald et al., 1994; Lehman et al., 1999). Briefly, wild-type cells and sro7 mutants were cultured to midlog phase in YPD medium and shifted for 2 h in YPD or YPD + 0.6 M NaCl media at 25°C. Harvested cells were spheroplasted and lysed, and a P3 membrane fraction enriched in post-Golgi vesicles was prepared by differential centrifugation (Walworth and Novick, 1987). This fraction was resuspended and layered onto a 20–40% sorbitol velocity gradient that was fractioned as described previously (Brennwald et al., 1994). The presence of the vesicle marker Snc1/2p and Ena1p-HA were determined by immunoblotting followed by quantitation on a LI-COR Odyssey Infrared imaging system (Cincinnati, OH).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The NaCl Sensitivity of the sro7 Mutant Is Due to Defective Ena1p Stability Rather than Defective ENA1 Expression
We have previously observed that mutants having a deleted SRO7 gene show increased sensitivity to NaCl (Larsson et al., 1998). As demonstrated in Figure 1, this sensitivity is strongly dependent on the pH of the growth medium. At acidic pH, the sro7 mutant showed wild-type growth behavior both in saline and nonsaline medium. At neutral pH, however, the presence of 0.5 M NaCl caused a significantly stronger growth inhibition of the mutant strain. S. cerevisiae uses two different transport systems to export Na⁺, the NHA1-encoded Na⁺/H⁺ antiporter (Banuelos et al., 1998) and the ENA1-encoded Na⁺-extruding ATPase (Garciadeblas et al., 1993; Wieland et al., 1995). Because Nha1p is active at low pH, whereas Ena1p has its optimum activity around neutral pH, we anticipated that ENA1 might be a prime target for the effects of the SRO7 deletion. We therefore analyzed ENA1 expression by Northern blot analysis (Figure 2A). Total RNA was extracted from wild-type and sro7 mutant cells at various time points after addition of NaCl to 0.5 M concentration to cultures growing exponentially in YPD medium. In wild-type cells there was a rapid, transient transcriptional induction of the ENA1 gene, reaching its maximum 30 min after the salt shock, followed by a return to low expression levels. The expression profile of the sro7 mutant was very similar to that of wild-type cells, indicating that the salt-sensitive phenotype is not a consequence of defective ENA1 expression.

View larger version (20K): [in this window] [in a new window]   Figure 1. Growth of wild-type and sro7 strains in the presence and in the absence of NaCl stress. E-flasks containing complete minimal CBS media supplemented with 0 or 0.5 M NaCl at pH 4.0 or pH 7.0 were inoculated from overnight cultures of wild-type (W303-1A) or sro7 (WKL-2A) cells to an OD610 of 0.05. Growth was monitored by following OD610.

View larger version (59K): [in this window] [in a new window]   Figure 2. The sro7 mutant shows wild-type expression of ENA1, and its salt tolerance is not improved by overexpression of ENA1. (A) Northern blot analysis of ENA1 expression after shift of exponentially growing wild-type (W303-1A) or sro7 (WKL-2A) strains to medium containing 0.5 M NaCl. Total RNA was extracted from cells and harvested at time points indicated. Each sample contains extract from the same number of cells. Transcript levels were first normalized to control mRNA (IPP1) and then presented relative the wild-type value at 30 min. (B) Drop-test monitoring growth of ena1 and ena1 sro7 mutants, carrying the pRS316 empty control plasmid and the same plasmid containing the ENA1 gene behind the strong PMA1 promoter (Wieland et al., 1995). Tenfold dilutions of CBS cultures of OD610 1.0 were spotted onto CBS and CBS + 0.5 M NaCl plates. Plates were photographed after incubation for 48 h at 30°C.

Having observed wild-type expression of ENA1 in sro7 mutants, we went on to examine the fate of the ENA1 encoded protein. The genomic copy of ENA1 in the wild-type and the sro7 mutant therefore was modified to encode three HA epitopes at the C-terminus of the protein (see Materials and Methods). Yeast cells expressing this tagged protein produced a protein detectable with HA antibodies on immunoblots, with an apparent molecular weight of 130 kDa. The tagged wild-type strain maintained its original salt tolerance, and the HA tagged sro7 mutant showed wild-type salt tolerance after reintroduction of the SRO7 gene on a plasmid, indicating that the tagged protein was functional in both strains (unpublished data). A first indication that lack of the SRO7 gene affects the stability of Ena1p came from immunoblot analysis of extracts of the sro7 mutant (Figure 3A). This analysis failed to detect any HA-tagged Ena1p 5 h after the mutant had been shifted to medium containing 0.5 M NaCl. In similar experiments with mutants transformed to contain the SRO7 gene on either a centromeric or a 2µ plasmid, tagged Ena1p was clearly detectable. We therefore performed a time-course study of the Ena1p fate in exponentially growing wild-type and sro7 strains shifted to medium of increased salinity. In wild-type cells the Ena1p levels increased after the shift to 0.5M NaCl, and after long-time incubation (12 h) there was a massive accumulation of Ena1p (Figure 3B). However, in sro7 mutants the Ena1p level initially increased as in wild-type cells, but then declined to undetectable levels >4 h after the salinity shift (Figure 3B). These observations suggest that maintained stability of Ena1p is dependent on a functional Sro7p.

View larger version (26K): [in this window] [in a new window]   Figure 3. The stability of Ena1p is dependent on SRO7. (A) Immunoblot analysis of Ena1p-HA expressing sro7 mutants transformed with and empty plasmid (pRS316), a CEN-SRO7 plasmid (pH/B316), and a 2µ-SRO7 plasmid (YEpH/B). Cells were cultured to midlog phase, exposed to 0.5 M NaCl for 5 h, harvested, and analyzed for the presence of Ena1p-HA. (B) A time-course study of the appearance of Ena1p-HA in wild-type and sro7 cells, after exposure to medium containing 0.5 M NaCl. Cell samples were withdrawn at stages indicated in the growth curve (arrows), and total protein extracts were analyzed for presence of Ena1p-HA. For immunoblot analysis protein extracts were subjected to SDS-PAGE in 10% polyacrylamide gels. All lanes were loaded with identical amounts of total protein. Blotted filters were analyzed for Ena1-HA by primary mouse anti-HA antibodies and secondary HRP-conjugated anti-mouse antibodies (Roche).

Ena1p Is Sorted in a MVE-dependent Way from Internal Compartments to the Vacuole in Salt-stressed sro7 Mutants
Most major pathways for degradation of membrane proteins terminate in the vacuole (Katzmann et al., 2002; Horak, 2003). To investigate the fate of Ena1p in the sro7 mutant, we therefore constructed the sro7 pep4 double mutant, in which vacuolar protease activity is diminished due to deletion of the gene for the master protease, Pep4p (Jones, 1991). If SRO7 deficiency results in sorting of Ena1p to the vacuole for degradation, the levels of Ena1p would be expected to remain unchanged in the sro7 pep4 mutant lacking active vacuolar hydrolases. This prediction was supported by immunoblot analysis (Figure 4A). After shift to increased salinity, the sro7 pep4 mutant maintained wild-type levels of Ena1p at time points when levels were clearly reduced in the sro7 single mutant. We conclude that Ena1p in salt-stressed sro7 mutants is diverted to the vacuole and subjected to Pep4p-dependent protease degradation.

View larger version (69K): [in this window] [in a new window]   Figure 4. Inactivation of the vacuolar protease Pep4p stabilizes Ena1p, but does not rescue growth of sro7 mutants on NaCl media. Time-course study of Ena1p-HA levels in (A) pep4, sro7 and pep4 sro7 mutants and (B) sro7, end4 sro7, sec15-1 sro7 and vps27 sro7 mutants after exposure of cells to medium containing 0.5 M NaCl. Cell samples were withdrawn at time points indicated, and total protein extracts were analyzed for presence of Ena1p-HA by immunoblot analysis. The sec15-1 sro7 (ts) mutant was incubated at 34°C during salt treatment to inactivate Sec15-1p without causing total growth arrest. Immunoblot analysis was performed as described in Figure 3 and Materials and Methods. (C) Drop-test monitoring growth of sro7, pep4, vps27, pep4 sro7, and vps27 sro7 on YP and YP + 0.7 M NaCl plates. Tenfold dilutions of YP cultures of OD610 1.0 were spotted onto the plates, which were photographed after incubation for 48 h at 30°C.

Missorting in sro7 Mutants Is Exacerbated by NaCl Stress
Despite the stabilization of Ena1p in the sro7 pep4 and sro7 vps27 mutants no obvious differences in salt tolerance were observed between these strains and the sro7 mutant (Figure 4C). Hence, stabilization of Ena1p was not sufficient per se to restore the salt tolerance of sro7 mutants. This suggests the cell is incapable of sorting Ena1p correctly to the plasma membrane in the absence of SRO7, even when Ena1p remains stable. This result agrees with the previous observation that overexpression of the ENA1 gene does not improve salt tolerance of the sro7 mutant (Figure 2B).

To further study the fate of Ena1p in wild-type cells and sro7 mutants, we used the pUG35-ENA1 plasmid (gift from M. Cyert) containing an ENA1-GFP insert driven by the methionine-repressible MET25 promoter. The expression of ENA1-GFP from this plasmid restores wild-type NaCl tolerance of ena1 mutants (unpublished data), demonstrating that the Ena1p-GFP fusion is functional. This construct allowed us to examine the influence of salt stress on Ena1p secretion, because ENA1 expression can be induced without NaCl exposure. Transformants of wild-type and sro7 cells cultured at repressing methionine concentration (0.5 mM) were washed free of methionine to de-repress ENA1-GFP expression and incubated in CBS medium with and without 0.6 M NaCl. Fluorescence microscopy of these transformants showed that Ena1p-GFP, independent of salinity, gradually accumulated in the periphery of wild-type cells, first appearing around the rim of the bud or in the bud neck of the cells (Figure 5A). A similar pattern was observed in nonstressed sro7 mutants. However, in sro7 mutants subjected to 0.6 M NaCl stress, the GFP staining was coincident with the lumen of the vacuoles, as visualized by costaining with the vital dye FM4-64, which stains the delimiting vacuolar membrane (Vida and Emr, 1995). These experiments confirmed routing of Ena1p to the vacuolar lumen in sro7 mutants and also demonstrate that the sorting into the endosomal pathway for vacuolar degradation is dependent on NaCl stress.

View larger version (50K): [in this window] [in a new window]   Figure 5. Mis-sorting of Ena1p in sro7 mutants is exacerbated by NaCl stress and is dependent on the ubiquitin ligase Rsp5p. (A) Wild-type cells and sro7 mutants transformed with the pUG35-ENA1 plasmid, expressing Ena1p-GFP from the MET25 promoter, were cultured overnight in CBS medium containing 0.5 mM methionine. These cultures were used to inoculate identical fresh medium, and cells were cultured to midlog phase before being stained for 15 min with the vital dye FM4-64, to visualize vacuolar membranes. The cells were then washed, resuspended in fresh medium, and incubated for another 1.5 h before being washed again and transferred to methionine-free medium with and without 0.6 M NaCl. Removal of methionine induces expression of Ena1p-GFP from the MET25 promoter. Samples were removed and photographed at the times indicated. (B) Wild-type cells, sro7 single mutants, and sro7npi1 double mutants, which express low levels of RSP5, were treated as in A and incubated for 2 h in methionine-free medium at 0.6 M NaCl. Yellow regions indicate Ena1p-GFP and FM4-64 colocalization.

sro7npi1

Genetic Interactions between SRO7 and Late Secretory Mutants
Lehman et al. (1999) reported that overexpression of SRO7 partially suppressed the temperature-sensitive phenotypes of mutations in SEC3, SEC8, SEC10, and SEC15, all of which encode subunits of the exocyst. However, high copy number suppression can also be the result of rather indirect interactions. We therefore tested if the sro7 knockout shows synthetic interactions with temperature-sensitive sec3-2, sec4-8, sec5-24, sec6-8, sec8-9, sec9-4, sec10-2, and sec15-1 mutations, by disrupting the SRO7 gene in the corresponding sec strains and testing growth at different temperatures. As shown in Figure 6, we saw clear evidence of synthetic interactions between sro7 and sec3-2, sec8-9, sec10-2, and sec15-1 at both 34 and 36°C, which, interestingly, are the same mutations that were suppressed by overexpression of SRO7 (Lehman et al., 1999). For sec4-8, we saw a very weak synthetic interaction with sro7 at 30°C, whereas no interactions were detected between sro7 and sec5-24, sec6-8, or sec9-4 (unpublished data). It should be noted, though, that the stronger phenotypes of these sec mutations, which prevent all growth at 34°C and above, make it more difficult to detect any synthetic interactions.

View larger version (107K): [in this window] [in a new window]   Figure 6. Genetic interaction between SRO7 and various SEC genes. The SRO7 gene was disrupted in various sec mutants, and temperature sensitivity was monitored at different temperatures. Top row represents growth of wild-type (NY179) and sro7 (H1394) strains at 22, 34, and 36°C. The following rows display growth of different sec single mutants (left column) and the corresponding sro7 sec double mutants (right column). Genetic interactions are revealed as increased temperature sensitivity of double mutants. Plates were photographed after incubation for 48 h at the indicated temperatures.

The Salt Sensitivity of sro7 Mutants Is Accompanied by a Late Secretory Defect and the Accumulation of Post-Golgi Secretory Vesicles

View larger version (51K): [in this window] [in a new window]   Figure 7. The sro7 mutant exhibits secretion defects and accumulation of post-Golgi vesicles at high salinity. (A). Secretion of Bgl2p. Assays were performed on isogenic wild-type and sro7 stains after a 2-h shift to YP medium with 0.6 M NaCl (+NaCl) or YP medium without added NaCl. The results show the internal accumulation of Bgl2p as a percentage of the total Bgl2 in the cell. (B) Electron microscopy of cells from WT and sro7 strains after a 2-h shift to YP medium with 0.6 M NaCl show that sro7 mutant cells accumulate 80–100-nm post-Golgi vesicles in the bud. This accumulation depends on the addition of salt, because no vesicles accumulate in sro7 cells in the absence of a salt stress. (C) Quantitation of the secretory vesicle accumulation in sro7 cells after a 2-h shift to 0.6 M NaCl/YP. Cells were scored for severity and penetrance of the secretory defects. The bar graph on the left shows the accumulation of vesicles in the indicated strains, expressed as vesicles per cell, and was determined by the number of 80–100-nm vesicles observed per cell per thin section. The graph on the right shows the penetrance of the vesicle accumulation expressed as the number of cells containing more than 10 vesicles per cell. A minimum of 100 cells were used for the quantitation of each strain.

To further characterize these vesicles as post-Golgi vesicles, velocity sedimentation analysis was performed. Lysates derived from spheroplasts of wild-type cells and sec6-4 and sro7 mutants having the chromosomal ENA1 gene modified to carry the 3xHA tag were layered onto sorbitol gradients as described previously (Adamo et al., 2001). All strains were shifted into 0.6 M NaCl media for 2 h to induce expression of Ena1p and the secretory defect associated with sro7 cells. As a positive control for a late secretory block, we shifted sec6-4 cells (containing Ena1-HA) into 0.6 M NaCl (to induce Ena1-HA) and then shifted the cells to 37°C for 2 h to impose the late secretory block. After centrifugation fractions were analyzed for Ena1p-HA, the vesicle marker Snc1/2p, and Bgl2p (unpublished data) by immunoblot analysis. The results, shown in Figure 8A (top graphs), strongly support the observations seen by EM and demonstrate sro7 mutants have a pronounced accumulation of post-Golgi vesicles (marked by Snc1/2p) in response to 0.6 M NaCl. We find these vesicles have a mobility (compare top graphs left and right) in the gradients that are identical to those that accumulate in sec6-4 cells, which is consistent with the EM and secretion results above. Finally we find that a peak of the periplasmic enzyme Bgl2p appears in the sro7 gradients (but not in wild-type gradient), which is coincident with the peak of Snc1/2p that appears in sro7 cells (unpublished data)—demonstrating that the accumulation of internal pools of Bgl2p in this mutant is due to a post-Golgi secretory defect.

View larger version (39K): [in this window] [in a new window]   Figure 8. (A) Accumulation of post-Golgi vesicles marked with Snc1/2p but not Ena1p in sro7 mutants shifted to high salt. Vesicle gradients of 20–40% sorbitol were performed on sro7 and isogenic wild-type strains after 2 h of growth in media with 0.6 M NaCl to induce both expression of HA-tagged Ena1 as well as the sro7 growth defect. As a positive control HA-Ena1p was introduced into a sec6-4 strain, which was grown in 0.6 M NaCl for 2 h (to induce HA-Ena1p) before shifting temperature to 37°C to induce the late secretory block. Normalized lysates from each strain were then layered onto the velocity gradients. Post-Golgi vesicles were detected by immunoblotting with rabbit anti-Snc1/2p antisera. HA-tagged Ena1p was detected with mAb 12CA5. Western blots were quantitated using an Odyssey InfraRed imaging system. (B) Ena1p is not retargeted to the vacuole after blocked exocytosis in late sec mutants. Wild-type (RSY255) cells and sec6-4 and sec4-8 mutants transformed with the pUG35-ENA1 plasmid, expressing Ena1p-GFP from the MET25 promoter, were cultured overnight in CBS medium and treated as described in legend to Figure 5 except that transferred cells were incubated at 25 or 37°C in the methionine-free medium with and without 0.6 M NaCl. Samples were removed and photographed 2 h after start of the final incubation.

We next examined the effect of Ena1p sorting by a late block in secretion by following the distribution of Ena1p-GFP in wild-type and late sec mutants. Wild-type (RSY255), sec4-8 and sec6-4 strains were transformed with the pUG35-ENA1 plasmid and incubated in basal medium at permissive (25°C) and restrictive (37°C) temperature after de-repression of ENA1-GFP expression (Figure 8B). Restrictive conditions clearly blocked delivery of Ena1p-GFP to the cell surface in the sec mutants, as opposed to the seemingly intact secretion at the permissive temperature. However, the blocked secretion did not result in any obvious vacuolar sorting of accumulated Ena1p-GFP, instead GFP fluorescence remained as a punctate haze in the cytosol, indicating stalled Ena1p traffic in the secretory system. A similar distribution of Ena1-GFP fluorescence was recorded for sec mutants when experiments were repeated at 0.6 M NaCl. Hence, a block at a late stage in exocytosis does not promote vacuolar targeting of Ena1p even when cells are exposed to high salinity.

We were unable to identify any obvious mis-targeting in NaCl-exposed sro7 mutants for a number of other GFP-tagged membrane proteins selected for analysis by microscopy. This analysis revealed an apparently correct targeting of the constitutively expressed sodium-proton anti-porter Nha1p in NaCl-stressed sro7 mutants (Figure 9). Similarly, a GFP-tagged version of Gap1p, expressed from the heterologous MET25 promoter, showed a distribution in NaCl-stressed sro7 mutants similar that of wild-type cells. Gap1p is thought to be carried by a class of post-Golgi vesicles distinct from the high-density Bgl2p carrying vesicles (Roberg et al., 1997). Similarly, other GFP tagged proteins examined, i.e., the polarly localized Sho1p and Ste2p, as well as the membrane associated Pbs2p (Stefan and Blumer, 1999; Reiser et al., 2000), maintained a typical wild-type-like distribution in sro7 mutants even during salt stress (unpublished data for the Ste2p and Pbs2p localization). We conclude that sro7 mutants exposed to salt stress accumulate post-Golgi vesicles and restrict Ena1p delivery to the plasma membrane, but that the surface delivery of other proteins examined is partly or insignificantly inhibited. Thus, loss of SRO7 affects transport of only specific proteins, which is in agreement with the observation that alternative pathways exist for the transport of membrane proteins to the plasma membrane (Harsay and Bretscher, 1995; Roberg et al., 1997; Harsay and Schekman, 2002).

View larger version (99K): [in this window] [in a new window]   Figure 9. Localization of selected GFP-tagged transmembrane proteins in wild-type and sro7 cells after a shift to high salinity. Cells were grown to midlog phase and then exposed to medium containing 0.5 M NaCl for 3 h before photography. Top, Nha1p-GFP, which localizes to the plasma membrane (Banuelos et al., 1998), shows a similar distribution in salt-stressed wild-type cells (left) and sro7 mutant cells (right). To express Nha1p-GFP, cells were transformed with the pGRU1 plasmid (Kinclova et al., 2001). Middle, Sho1p-GFP displays a similar pattern of polarized localization in NaCl-stressed wild-type and sro7 mutant cells, being concentrated in the growing bud and at the mother-bud neck as previously reported (Reiser et al., 2000). Bottom, Gap1p-GFP expressed from the MET25 promoter in the pUG35-GAP1 plasmid exhibits similar distribution in salt-stressed wild-type and sro7 mutant cells. Gap1p shows distribution to ER and the plasma membrane as reported for wild-type cells growing under nonstress conditions (Roberg et al., 1997).

Overexpression of the RSN1 Gene Suppresses Salt Sensitivity of the sro7 Mutant

View larger version (75K): [in this window] [in a new window]   Figure 10. The RSN1 gene rescues growth of the sro7 mutant at high salinity. Cultures of wild-type (BY 4741), rsn1, sro7, and ena1 mutant strains transformed with the 2µ plasmid pRSN1 or the empty vector pRS423 were spotted in 10-fold dilutions onto defined CBS medium with or without NaCl as indicated, the first spot representing OD610 0.1. Plates were incubated for 48 h at 30°C before photography.

View larger version (33K): [in this window] [in a new window]   Figure 11. Rsn1p is a plasma membrane protein and multicopy expression of RSN1 reroutes Ena1p to the cell surface in NaCl-stressed sro7 mutants. (A) Transmission image and fluorescence intensity distribution of CFP-tagged Rsn1p in wild-type cells as analyzed by confocal microscopy. The graph shows a line profile of the fluorescence intensity measured along the black line indicated in the figure. The tagged protein is clearly concentrated in the bud neck region (corresponding to the peak at 6 µm). (B) Subcellular fractionation of cells expressing Rsn1p-CFP. Total protein extract was centrifuged at 10,000 x g and 100,000 x g to separate soluble cytosolic proteins from pelletable structures. P10, 10,000 x g pellet; P100, 100,000 x g pellet; S100, supernatant from 100,000 x g centrifugation. (C) Rsn1p-CFP localizes to a membrane fraction. Cell lysates were treated with 0.1 M KCl, 1.0 M KCl, or 1% Triton X-100. Pellet and supernatant fractions were then separated by centrifugation at 100,000 x g. Rsn1p-CFP was detected by SDS-PAGE followed by immunoblotting using an anti-GFP antibody. (D) Immunoblot analysis of Ena1p-HA levels in sro7 mutants containing a control plasmid or a multicopy RSN1 plasmid. Cells were grown to exponential phase and shifted to medium containing 0.6 M NaCl before sampling at indicated time points. Immunoblot analysis was preformed as described in Figure 3 and Materials and Methods. Total proteins were extracted by alkaline cell lysis, and Ena1p-HA was detected by SDS-PAGE and immunoblotting with anti-HA antibody. Each sample represents an equal amount of total protein. (E) Fluorescence images of sro7 mutants transformed with the pUG35-ENA1 plasmid expressing Ena1p-GFP from the MET25 promoter and the pRS423 empty vector (left) or the pRS423-RSN1 vector (right) Cells cultured on defined medium containing 1 mM methionine and washed before transfer to methionine-free 0.5 M NaCl medium. Photographs were taken after 3-h incubation in the saline medium.

Increased Expression of RSN1 Enhances Ena1p Stability in sro7 Mutants and Retargets the Protein to the Plasma Membrane

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Previous work has shown that deletion of the yeast SRO7 gene produces a NaCl-sensitive phenotype and a disrupted Na⁺/K⁺ balance in cells subjected to NaCl stress (Larsson et al., 1998). Here we demonstrate that the NaCl sensitivity of sro7 mutants is due to defective targeting of the ENA1 gene product to its final destination in the plasma membrane. The ENA1 gene encodes a salinity-induced sodium transporter that belongs to the family of ion-transporting P-type ATPases and constitutes the main pathway for sodium export in yeast (Haro et al., 1991; Haro et al., 1993; Wieland et al., 1995). In cells lacking Sro7p the salt-induced sodium pump becomes degraded before the arrival at the plasma membrane, because additional mutations in the sro7 background that blocked Golgi to plasma membrane transport (sec15) or inhibited internalization from the cell surface (end4), did not prevent Ena1p degradation. The loss of Ena1p in sro7 mutants is caused by retargeting to the vacuole (Figures 4A and 5), probably via the MVE pathway because degradation of Ena1p does not occur in sro7 vps27 mutants (Figure 4B). Vps27p mediates, in conjunction with the ESCRT complexes (endosomal sorting complex required for transport), sorting of ubiquitinylated membrane proteins destined for degradation in the vacuole (Bilodeau et al., 2002; Katzmann et al., 2003