Golgi-to-Endoplasmic Reticulum (ER) Retrograde Traffic in Yeast Requires Dsl1p, a Component of the ER Target Site that Interacts with a COPI Coat Subunit

Media, Strains, Plasmids, and Antibodies

The Escherichia coli strain XL1-Blue (Stratagene, La Jolla, CA), which was used throughout this work, was maintained on standard media (Miller, 1972) and transformed by the Hanahan method (Hanahan, 1983). Saccharomyces cerevisiae strains, described in Table 1, were maintained either on rich media (YPD) containing 1% Bacto-yeast extract, 2% Bacto-peptone, 2% glucose, and 20 μg/ml adenine sulfate or on synthetic complete media (SC) containing 0.67% yeast nitrogen base without amino acids, 2% glucose, and the appropriate amino acid supplement (Rose et al., 1990), unless otherwise noted. Yeast transformations were by the method of Schiestl and Gietz (1989). Yeast strains were maintained at 30°C unless otherwise noted, and all incubations designated as 23°C were completed at room temperature, which ranged from ∼19°C to 23°C. All experiments used log phase cultures with an OD₆₀₀ between 0.5 and 1.0. Diploid strains were sporulated by incubation for 2 to 4 d at 23°C in liquid media consisting of 1% KOAc and 0.02% glucose.

To obtain dsl1 mutant strains used in the mating assay (Figure 3), strains GWY379 and GWY380 were constructed by mating GWY230 and GWY233, respectively, with PC13 containing pBR15. After sporulation of diploids and dissection of tetrads, the temperature-sensitive MATα, Ura⁺, Leu⁺, His⁻, Lys⁻ spores were isolated. The strains were backcrossed once again to obtain the temperature-sensitive MATa, Ura⁺, Leu⁺, His⁻, Lys⁻ segregants used in the mating assay.

Plasmid pBR4 was constructed by excising the 3.0-kb XhoI/NotI fragment containing full-length DSL1 from pSC2 and ligating it into similarly digested pRS415. Plasmid pBR15 was generated by inserting a 4.3-kb BamHI fragment containing STE2 from MR3264 (kindly provided by M. Rose, Princeton University, Princeton, NJ) into BamHI digested pRS413. To generate the plasmid expressing GST-Dsl1p, pSV59, the complete DSL1 open reading frame was amplified by polymerase chain reaction (PCR) placing EcoRI sites upstream of the start codon and downstream of the termination codon with the use of primers D1 (5′ CGG-AAT-TCA-TGG-AGT-CTC-TTT-TTC-C 3′) and D2 (5′ CGG-AAT-TCA-TGT-AAC-CTA-TCC-TAC-G 3′). The PCR product was then digested with EcoRI and ligated into a similarly digested vector, pGEX4T-1 (Amersham Pharmacia Biotech, Piscataway, NJ). To generate pBR2, pSV59 was digested with EcoRI and the liberated fragment was ligated into similarly digested pGBDU-C1 (kindly provided by P. James, University of Wisconsin, Madison WI) (Table 2). pSV91 was generated by digestion of pSV59 with BamHI and SmaI and ligation of the liberated fragment into a similarly digested pQE31 (QIAGEN, Santa Clarita, CA). Because the BamHI site used is 51 base pairs downstream from the first base pairs in the open reading frame, this construct expresses a His₆-tagged Dsl1p fragment that lacks its first 17 amino acid residues. Plasmid pSV57 contains full-length DSL1 with a NheI site before the termination codon and was generated by PCR with primers that place EcoRI sites upstream of the start codon and downstream of a primer-encoded NheI site and termination codon (D1 as described above and D3 [5′ CG-GAA-TTC-TTA-GCT-AGC-ATC-ATC-TAG-AGC-AGT-GC]). The PCR product was digested with EcoRI and ligated into similarly digested pRS416. To generate pSV61, a 100-base pair fragment encoding the triple hemagglutinin (HA) epitope sequence was removed from plasmid MR2654 (kindly provided by M. Rose) by digestion with XbaI and ligated into NheI digested pSV57. pSV66 was constructed by subcloning the HindIII/SalI fragment containing OCH1-3xHA under the control of the OCH1 promoter from pOH (Harris and Waters, 1996) into similarly digested pRS415.

GST-Dsl1p was expressed from plasmid pSV59, purified as per Amersham Pharmacia Biotech instructions, and used to inoculate rabbits by standard procedures (Harlow and Lane, 1988). The His₆-Dsl1p fragment was expressed from pSV91, purified according to standard procedures under nondenaturing conditions (QIAGEN), and covalently linked to cyanogen bromide-activated Sepharose according to the manufacturer's instructions (Amersham Pharmacia Biotech). The resulting His₆-Dsl1p-conjugated Sepharose was used to affinity purify antibodies against Dsl1p by standard procedures (Harlow and Lane, 1988). Yeast extracts used for the characterization of the affinity-purified α-Dsl1p antibody were generated as described (Ohashi et al., 1982).

Antibodies that recognize carboxypeptidase Y (CPY), Kar2p, and Sec61p were the kind gifts of Tom Stevens (University of Oregon, Eugene, OR), Mark Rose (Princeton University, Princeton, NJ), and Randy Schekman (University of California, Berkeley, CA), respectively. Anti-invertase and anti-Sed5p (Lupashin and Waters, 1997) were generated in this laboratory by standard methods. Anti-phosphoglycerate kinase (PGK) was from Molecular Probes (Eugene, OR).

Metabolic Labeling and Immunoprecipitation of CPY and Invertase

For pulse-chase experiments, log phase strains (RSY255, GWY270, MS1554, GWY230, and GWY233) were shifted to SC-methionine media that contained 2% raffinose and 0.1% glucose (to induce the expression of invertase) for 1 h before the temperature shift. One OD₆₀₀ unit of cells was shifted to the appropriate temperature by centrifugation and resuspension in 0.5 ml of the same prewarmed media. After incubation at the same temperature for 20 min, cells were metabolically labeled with 50 μCi of Tran³⁵S-label (ICN Pharmaceuticals, Irvine, CA) for 10 min. For the chase, 10 μl of 250 mM cysteine, 250 mM methionine was added and incubation was continued for 20 min. Processing was stopped by placing the cells on ice and adding 20 μl of 10 mg/ml cycloheximide, 500 mM NaN₃. CPY or invertase was immunoprecipitated as previously described (Sapperstein et al., 1996) with 2 μl of α-CPY antibody or 5 μl of α-invertase antibody (this laboratory) per OD₆₀₀ unit of cells. The immunoprecipitated material was analyzed by SDS-7% PAGE and autoradiography.

Analysis of Kar2p Secretion

Log phase strains (RSY255, GWY230, and GWY233) containing vector (pRS416) or CEN DSL1 (pSC2) were spotted onto YPD plates that, after the media were adsorbed into the plates, were overlayed with nitrocellulose prewet in sterile water. After a 16-h incubation, the nitrocellulose was removed and the levels of Kar2p were analyzed by immunoblotting with a 1:100,000 dilution of α-Kar2p antibody and ECL PLUS detection (Amersham Pharmacia Biotech).

Mating Assay

The mating assay is essentially as described by Letourneur et al. (1994). Briefly, log phase PC13, PC75, GWY379, and GWY380 strains were grown on YPD plates, each strain was replica plated to five YPD plates, and the replicas were incubated overnight at 23°C. One plate from each strain was then incubated for 2 h at 23, 27, 30, 34, or 37°C before replica plating onto lawns of RSY255, the mating tester strain, which had been grown on YPD. After 6 h at the same temperature to allow mating, cells were replica plated to SC-Ura-Lys and incubated at 23°C for 2 d to allow growth of diploids.

Synthetic Lethality

The appropriate mating type of strains dsl1-4 (GWY230 or GWY415) and dsl1-7 (GWY233 or GWY414) were mated with EGY101 (ret1-1), CKY100 (sec27-1), PC133 (ret2-1), RSY277 (sec21-1), MY7512 (ufe1-1), PC137 (tip20-5), and RSY275 (sec20-1). Resulting diploids were sporulated and dissected as described above. Segregants of each tetrad were replica plated to YPD and plates were incubated at 23, 27, 30, 34, or 37°C for 2 d. Putative tetratypes were identified as those sets of spores displaying 1:3 viable:inviable ratio at 37°C, because all single temperature-sensitive mutants are inviable at 37°C. Genotypes of all the spore-derived colonies in the putative tetratypes were determined by complementation of temperature sensitivity with plasmids containing DSL1, SEC20, or TIP20 (pSC2, 2 μm SEC20, or pTM2, respectively). Cotransformation with plasmids pBR4 and 2 μm SEC20 were used for complementation of the dsl1-7 sec20-1 double mutant. The dsl1-7 tip20-5 double mutant was inviable at all temperatures; therefore, its genotype was inferred from the other segregants.

Subcellular Fractionation and Membrane Extraction

Subcellular fractionation and membrane extraction were performed as previously described (VanRheenen et al., 1998). Briefly, a log phase 250 ml RSY255 culture was grown in YPD, harvested, and resuspended at 30 OD₆₀₀/ml in Buffer 88 [20 mM HEPES, pH 7.0, 150 mM KOAc, 5 mM Mg(OAc)₂] containing 1 mM dithiothreitol (DTT) and 1× protease inhibitor mix (2 μg/ml aprotinin, 0.5 μg/ml leupeptin, 4 mM PEFABLOC, 2 μM pepstatin A, and 0.5 mM phenanthroline), and lysed by vortexing in the presence of 0.3 g of 425–600 μm acid-washed glass beads (Sigma, St. Louis, MO). This crude lysate was centrifuged twice at 1000 × g and the second supernatant was diluted to a protein concentration of ∼5 mg/ml with Buffer 88. This clarified lysate was then centrifuged at 175,000 × g for 60 min in a TLA100.2 rotor (Beckman Instruments, Palo Alto CA), generating the supernatant (S) or pellet (P) fractions. Supernatant and pellet fractions, derived from equivalent amounts of starting material, were resolved by SDS-12%PAGE and analyzed by immunoblotting with antibodies against Dsl1p, Sed5p, Sec61p, and PGK (Figure 5B).

Extractions were performed on Dsl1p-containing total yeast membranes that had been isolated on a step gradient. Lysate (2 ml), generated as described above, was layered onto a step gradient composed of 2 ml of Buffer 88, 1 mM DTT, 50% sucrose, and 8 ml of Buffer 88, 1 mM DTT, 10% sucrose. The gradient was centrifuged at 40,000 × g for 2 h at 4°C in a Beckman SW41 rotor and membranes were collected from the 10%/50% sucrose interface. Membranes (100 μl/reaction) were mixed with an equal volume of 2× extraction buffer and then diluted to 1 ml in 1× extraction buffer, resulting in the following final concentrations of extraction reagent: 1% Triton X-100; 1 M NaCl; or 0.1 M Na₂CO₃, pH 11. In addition, a mock extraction was performed in which 100 μl of membranes was diluted to 1 ml in Buffer 88. After a 45-min incubation on ice, 800 μl of each sample was layered over a 200-μl 10% sucrose cushion made in the appropriate 1× extraction buffer and centrifuged at 175,000 × g for 60 min at 4°C in a Beckman TLA100.2 rotor. The top 900 μl of the solution was removed as the supernatant fraction and the remaining 100 μl, including a visible pellet, was resuspended in an equivalent volume (900 μl total) of Buffer 88. Samples were then resolved by SDS-10% PAGE and analyzed by immunoblotting with affinity-purified anti-Dsl1p and anti-Sed5p antibodies (Figure 5C).

Sucrose Density Gradients

Subcellular fractionation (Figure 6A) was based on the method of Antebi and Fink (1992) with the following modifications. Log phase RSY255 containing pSV66 cells were grown in 2 liters of SC-Leu, harvested by centrifugation, resuspended at 100 OD₆₀₀ units/ml in 100 mM Tris-Cl, pH 9.4, 10 mM DTT, and incubated at room temperature for 10 min. Cells were centrifuged and resuspended at 100 U/ml in 0.7 M sorbitol, 10 M Tris-Cl, pH 7.4, 0.75% yeast extract, 1.5% Bacto peptone, 0.5% glucose, and incubated at 30°C for 45 min in the presence of 0.5 mg/ml Zymolyase 100T (Seikagaku, Tokyo Japan). Spheroplasts were centrifuged at 4000 rpm for 5 min at 4°C in an SA600 rotor and then resuspended in 1 ml of lysis buffer 1 (10 mM HEPES, pH 7.4, 12.5% sucrose, 1 mM EDTA, 1× protease inhibitor mix). Spheroplasts were gently lysed by 10 hand strokes with a 5-ml Dounce homogenizer. Lysate was centrifuged at 3000 rpm and the supernatant was transferred to a fresh tube. The unlysed cell pellet was resuspended in 1 ml of lysis buffer 1 and homogenization and centrifugation was repeated. Supernatants were pooled and 1 ml of lysate was layered onto gradients containing 1-ml steps of 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, and 60% sucrose (wt/vol) in 10 mM HEPES, pH 7.4, 1 mM MgCl₂. Gradients were centrifuged in an SW41 rotor (Beckman Instruments) at 38,000 rpm (174,000 × g) for 2.5 h at 4°C after which twelve 0.8-ml fractions were collected from the top of the gradient. Aliquots from each fraction (0.5% total) were analyzed by SDS-12% PAGE, immunoblotted with anti-HA, anti-Kar2p, or anti-Dsl1p, and visualized by ECL PLUS.

Indirect Immunofluorescence

Log phase GWY199 and GWY416 cells (8 ml) were centrifuged at 15,000 rpm for 5 min and the cell pellet was resuspended in 2 ml of 4% paraformaldehyde, 0.1 M KH₂PO_4, pH 6.5, 1 mM MgCl₂ (prepared as follows: 2 g of paraformaldehyde was heated to 55°C in 45 ml of water, 210 μl of 10 N NaOH was added to solubilize the paraformaldehyde, 0.68 g KH₂PO₄ and 50 μl 1 M MgCl₂ were added, the volume was brought to 50 ml, and the solution was cooled to room temperature). Cells were incubated in fixation solution at 23°C overnight with rocking. Fixed cells were washed twice with 1-ml aliquots of spheroplasting buffer (0.1 M KH₂PO_4, pH 6.5, 1.2 M sorbitol) and then spheroplasts were generated in 1 ml of 0.5% β-mercaptoethanol, 2.5% glusulase (NEN), 0.15 mg/ml Zymolyase 100T in spheroplasting buffer for 1 h at 30°C followed by washing with 1-ml aliquots of 1.2 M sorbitol in phosphate-buffered saline (PBS). Cells were then incubated in 2% SDS, 1.2 M sorbitol for 10 min at 23°C, and then washed twice in 1.2 M sorbitol in PBS. Subsequent, methanol/acetone fixation, blocking, incubation with 1:2000 anti-HA or anti-Kar2p, incubation with 1:2000 Alexa Fluor 568 goat anti-mouse or Alexa Fluor 488 goat anti-rabbit secondary antibodies (Molecular Probes), diamidophenylindole staining, and fixation were done as described (Adams et al., 1998).

Size Exclusion Chromatography and Complex Immunoprecipitation

Log phase RSY255 cells, grown in 500 ml of YPD, were harvested by centrifugation, resuspended at 100 OD₆₀₀ units/ml in 100 mM Tris-Cl, pH 9.4, 10 mM DTT and incubated at room temperature for 10 min. Cells were centrifuged and resuspended at 100 OD₆₀₀ units/ml in 0.7 M sorbitol, 10 mM Tris-Cl, pH 7.4, 0.75% yeast extract, 1.5% Bacto peptone, 0.5% glucose, and incubated at 30°C for 45 min in the presence of 0.5 mg/ml Zymolyase 100T. Spheroplasts were centrifuged at 4000 rpm for 5 min in an SA600 rotor and then resuspended at 10 OD₆₀₀ units/ml in lysis buffer 2 (20 mM HEPES, pH 7.4, 50 mM KOAc, 2 mM EDTA, 0.1 M sorbitol, and 1× protease inhibitor mix). Spheroplasts were lysed on ice with 20 strokes of a Sorvall Omni-mixer set at the maximum speed. The lysate was cleared twice by centrifugation at 3000 rpm for 5 min in an SA600 rotor. The cleared supernatant was transferred to a new tube that was centrifuged at 13,500 rpm in an SA600 rotor for 15 min. The membrane pellet was washed twice in 1 ml of Buffer 88 through gentle pipetting and resuspended in 1 ml of Buffer 88 with a 2-ml Dounce homogenizer. For size exclusion chromatography (Figure 7A), aliquots (150 μl) of washed membranes were incubated on ice in Buffer 88 containing either 1% n-dodecyl-β-d-maltoside or 1% Triton X-100H in a total volume of 1 ml for 1 h. An 800-μl sample was overlayed on a 200-μl cushion of Buffer 88 and either 10% (wt/vol) sucrose, or 1% n-dodecyl-β-d-maltoside and 10% (wt/vol) sucrose, or 1% Triton X-100H and 10% (wt/vol) sucrose, and then centrifuged in a TLA100.2 rotor at 100,000 × g (48,000 rpm) for 1 h at 4°C. The supernatant was removed and 50 μl was chromatographed on a Superose 6 PC3.2/30 column at a flow rate of 50 μl/min with the use of a SMART System (Amersham Pharmacia Biotech). Fractions (75 μl) were collected starting just before the void volume, precipitated with trichloroacetic acid, and analyzed by SDS-10% PAGE and immunoblotting with anti-Dsl1p antibodies and ECL PLUS development.

For immunoprecipitation of the Dsl1p complex (Figure 7B), RSY255 and GWY199 membranes were prepared as described for size exclusion chromatography, except that the starting volume was 2 liters and the membrane pellet was resuspended in 1 ml of Buffer 88 containing 3% Triton X-100H and incubated on ice for 45 min to extract Dsl1p from membranes. Aliquots (800 μl) were placed on 200-μl cushions composed of Buffer 88, 10% (wt/vol) sucrose, 3% Triton X-100H and centrifuged in a TLA100.2 rotor at 100,000 × g for 60 min at 4°C. Immunoprecipitation reactions consisted of 750 μl of the supernatant, 250 μl of IP buffer 1 (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% TX-100H, 2 mM NaN₃) and 50 μl of 50% anti-HA.11-conjugated protein G-Sepharose beads (Covance, Denver, PA). After an overnight incubation with rocking at 4°C, the beads were washed four times with 1-ml aliquots of IP buffer 1. Thirty-five microliters of 2% SDS was added to the beads, bound protein was eluted by incubation at 100°C for 5 min, and 30% of the eluates were resolved by SDS-12% PAGE and visualized by silver staining.

Coimmunoprecipitation of Tip20p-myc and Dsl1p

Log phase RSY255 cells containing pTM2 (75 OD₆₀₀ units), grown in SC-Ura, were washed once with 20 mM Tris-Cl, pH 7.5, 20 mM NaN₃ and resuspended in 5 ml of IP buffer 2 (50 mM HEPES, pH 7.4, 150 mM KCl, 1 mM EDTA, 1 mM DTT, 1× protease inhibitor mix). The cultures (1.5-ml aliquots) were transferred to 2-ml screw cap conical tubes containing 1 g of 425–600-μm acid washed glass beads and lysed in a Biospec Products Mini-beadbeater-8 at full power for 4 min at 4°C. The supernatant was collected after a 15-min centrifugation at 15,000 rpm at 4°C and incubated with 50 μl of 50% slurry of protein A-Sepharose/IP buffer 2 containing 1% Triton X-100 in a final volume of 1.2 ml for 1 h at 4°C with rocking. This served to remove components that nonspecifically bind to protein A-Sepharose, and to detergent extract Dsl1p from membranes. After a 15-min centrifugation at 15,000 rpm at 4°C in a microcentrifuge, 1-ml aliquots of the supernatant were transferred to 1.5-ml tubes containing 60 μl 50% protein A-Sepharose beads conjugated with anti-myc antibody (9E10) and either 5 μl of PBS or 5 μl of 200 μg/ml myc peptide (EQKLISEEDL) in PBS. A separate 1-ml aliquot was incubated without beads to serve as a control for total protein. After 2 h with rocking at 4°C, samples were centrifuged at 15,000 rpm for 30 s. Beads were washed four times with 1 ml of IP buffer 2, and the supernatants and unfractionated control sample were precipitated with trichloroacetic acid. Samples were analyzed by SDS-10% PAGE, immunoblotted with anti-Dsl1p and anti-Sec61p antibodies, and visualized with ECL PLUS.

Yeast Two-Hybrid Screen

The yeast two-hybrid screen (Figure 8) was exactly as described by James et al. (1996) except that PJ69–4A yeast strain carried the bait plasmid pBR2, which encodes a Gal4p-DNA binding domain-Dsl1p fusion protein.

DSL1 Mutants Exhibit Defects Characteristic of Retrograde Trafficking under Conditions Where No Anterograde Trafficking Defects Are Apparent

In our previous work (VanRheenen et al., 2001) we showed that some dsl1 mutants could be genetically suppressed by overexpression of SEC21, which encodes the γ-subunit of the COPI retrograde coat. This finding stimulated us to further explore the possibility that Dsl1p is involved in retrograde Golgi-to-ER traffic. The phenotypes we had previously observed, including the block in ER-to-Golgi traffic, were consistent with this possibility. Indeed, many of the components that are known to function in retrograde Golgi-to-ER traffic were originally characterized as mutants that impact on anterograde ER-to-Golgi traffic (Novick et al., 1980). It is presumed that these anterograde defects are secondary consequences of a defect in retrograde traffic (Cosson et al., 1997; Gaynor and Emr, 1997).

Several mutants that block retrograde traffic exhibit a defect in ER-to-Golgi traffic of CPY under conditions where invertase traffics normally (Gaynor and Emr, 1997). Although the reason for this is not clear it has been proposed that the cause may be failure to recycle a component(s) required for ER egress of CPY. To determine whether dsl1-4 and/or dsl1-7 mutants exhibit a similar phenotype we performed pulse-chase analysis of CPY and invertase traffic at a range of temperatures from 23 to 37°C (Figure 1). CPY undergoes an ordered series of glycosylation and proteolytic events en route to the vacuole that results in characteristic changes in molecular mass: the p1 form (67 kDa) has yet to enter the Golgi, the p2 form (69 kDa) has received Golgi-specific glycosyl modifications, and the mature form (61 kDa) has been proteolytically processed in the vacuole (Stevens et al., 1982). Similarly, invertase is post-translationally modified as it moves from the ER, where it is present in distinct core-glycosylated forms, to the Golgi, where the oligosaccharides are extended by the addition of mannose residues leading to a population of molecules with varying molecular masses that migrate as a “smear” on SDS-PAGE.

We found (Figure 1) that both CPY and invertase traffic ceased simultaneously between 34 and 37°C in the dsl1-4 strain (compare lanes 7 and 12), providing no support for a retrograde defect. However, in the dsl1-7 strain, CPY traffic was compromised at a lower temperature (30°C, compare lanes 5 and 15) than invertase traffic (34°C, compare lanes 6 and 16). Thus, under at least one condition, dsl1-7 exhibits a defect in CPY traffic, whereas invertase traffic is unaffected. This is not the expectation for a component involved (exclusively) in anterograde traffic and has been reported previously for other retrograde trafficking mutants (Gaynor and Emr, 1997).

One of the hallmarks of mutants in Golgi-to-ER retrograde traffic is the inability to recycle ER-resident proteins, such as the chaperone Kar2p, that have escaped from the ER. To examine whether dsl1 mutants exhibit this phenotype, wild-type, dsl1-4, and dsl1-7 strains containing a control low-copy vector, or the same vector bearing DSL1, were grown at 37°C for 16 h in contact with nitrocellulose, which binds secreted Kar2p and allows subsequent immunodetection (Figure 2). We found that neither dsll1 mutant secretes Kar2p at the permissive temperature (our unpublished results), but that both mutants secrete the protein at the restrictive temperature. Control experiments indicate that Kar2p is not released due to cell lysis (our unpublished results). Last, as a control to address the possibility that Kar2p secretion in the dsl1 mutants might be due to increased cellular levels of Kar2p, perhaps exceeding the capacity of the Golgi-ER recycling system (Belden and Barlowe, 2001), intracellular levels of Kar2p were examined by immunoblotting total cell extracts. We found that, compared with wild-type cells, Kar2p levels in the mutants were not affected significantly at the permissive or restrictive temperature (our unpublished results); thus, Kar2p induction is unlikely to account for the Kar2p secretion.

To further probe this possibility that Dsl1p plays a role in retrograde Golgi-to-ER traffic, we examined the effect of dsl1 mutations in an established system that is sensitive to defects in retrograde Golgi-to-ER traffic (Letourneur et al., 1994). In this assay, the α-factor receptor Ste2p, which must be on the cell surface to allow mating, is retained in the ER by virtue of its fusion to the ER-resident protein Wbp1p, which displays a cytoplasmic dilysine retention signal. The ste2Δ STE2-WBP1 strain is sterile due to retrieval of Ste2p-Wbp1p to the ER, but it can mate if certain retrograde Golgi-to-ER transport components are mutated, thereby allowing the α-factor receptor to be transported to the cell surface (Letourneur et al., 1994; Cosson et al., 1997). We constructed dsl1-4 ste2Δ STE2-WBP and dsl1-7 ste2Δ STE2-WBP strains and tested them, along with positive and negative control strains, for their ability to mate at a range of temperatures. The strains (which are Ura⁺Lys⁻) were grown for 2 h, replica plated to lawns of wild-type cells of the opposite mating type (which are Ura⁻Lys⁺), and incubated for 6 h at temperatures ranging from 23 to 37°C to allow an opportunity to mate. These plates were then replica plated to medium selective for diploids (Ura⁺Lys⁺) and incubated for 2 d.

As expected, the ste2Δ STE2-WBP control strain does not mate at any temperature (Figure 3, first row). In contrast, the ret1-1 ste2Δ STE2-WBP strain, which bears a mutation in the α-COP subunit of the COPI coat that disrupts recognition of the retrieval signal without compromising retrograde traffic in general (Letourneur et al., 1994), mates at all temperatures (Figure 3, second row). Interestingly, both the dsl1 strains display temperature-dependent mating. Robust mating ability was conferred to dsl1-4 at its semipermissive temperature of 34°C (Figure 3, third row). At the restrictive temperature of 37°C, only a small population of dsl1-4 cells is able to mate. Similarly, dsl1-7 was able to mate at the lowest restrictive temperatures of 30°C, but not at the restrictive temperatures of 34 or 37°C (Figure 3, fourth row).

These findings suggest that the Ste2p-Wpb1p fusion protein is maintained in the ER in dsl1 mutants at the permissive temperature, which precludes mating. As the temperature is increased to the semipermissive temperature, enough fusion protein escapes to enable mating. Further increases in temperature into the restrictive range may preclude mating due to the fact that anterograde transport, which is required to deliver the fusion protein to the cell surface, is blocked (Figure 1).

Taken together, the biochemical data (Figures 1 and 2) and the mating assay (Figure 3) suggest that Dsl1p is required for retrograde Golgi-to-ER traffic. An additional role in ER-to-Golgi traffic remains a possibility.

Genetic Interactions of DSL1 with Genes Encoding Retrograde Transport Components

Because Dsl1p appeared to be involved in retrograde transport, we sought to identify synthetic lethal interactions between dsl1 mutants and mutants in other genes that play a role in this process. We therefore examined the growth phenotypes of dsl1-4 and dsl1-7 strains bearing mutations in retrograde COPI coat components α-COP, β′-COP, γ-COP, or δ-COP (ret1-1, sec27-1, sec21-1, or ret2-1, respectively), the retrograde t-SNARE Ufe1p (ufe1-1), or the Ufe1p-associated proteins Sec20p and Tip20p (sec20-1 and tip20-5).

We found no synthetic interactions between the dsl1 mutants and ret1-1, sec27-1, ret2-1, sec21-1, or ufe1-1 (our unpublished results). In contrast, both dsl1-4 and dsl1-7 exhibited synthetic lethal interactions with sec20-1 and tip20-5. Two representative tetratypes from dissection of the dsl1-7/DSL1 sec20-1/s20 diploids (Figure 4A) and dsl1-7/DSL1 tip20-5/TIP20 diploids (Figure 4B) grown at 23, 27, and 37°C are shown. The dsl1-7 sec20-1 double mutant is inviable at the dsl1-7 semipermissive temperature of 27°C (Figure 4A) and dsl1-7 tip20-5 double mutant is inviable at all temperatures tested (Figure 4B). This synthetic lethality provides further support for a retrograde role for Dsl1p, and suggests that it may act at the ER target site in conjunction with Sec20p and Tip20p.

In contrast to the clear synthetic lethal interactions, we found no suppression of dsl1-4 (GWY230) or dsl1-7 (GWY233) by overexpression of TIP20 or SEC20. Similarly, we found no suppression of sec20-1 (RSY275) or tip20-5 (PC137) by overexpression of DSL1 (our unpublished results).

Dsl1p Is a Peripheral Membrane Protein of Endoplasmic Reticulum

To initiate biochemical analyses of Dsl1p, we generated polyclonal α-Dsl1p antisera in rabbits. After affinity purification, the antibodies recognized a single protein in crude yeast extracts that migrated at ∼88 kDa (Figure 5A, lanes 1 and 3), the predicted molecular mass of Dsl1p. Transformation of a wild-type strain with a high-copy plasmid bearing DSL1 resulted in the overexpression of this protein (Figure 5A, lane 2). Examination of the original mutant found in the DSL screen (VanRheenen et al., 2001), dsl1-1, revealed that the full-length protein was absent; rather, a protein migrating at ∼75 kDa was observed, and this truncated protein was less abundant than wild-type Dsl1p (Figure 5A, lane 4). These data demonstrate that the antibody specifically recognizes Dsl1p.

Many cytosolic proteins that function in the secretory pathway have membrane-associated pools. To investigate whether this is the case for Dsl1p, we centrifuged a yeast lysate at 175,000 × g to separate cytosolic from membrane-associated proteins, and probed the supernatant (S) and pellet (P) fractions for Dsl1p by immunoblotting. Almost all of the Dsl1p was contained in the pellet fraction, as were the integral membrane proteins Sed5p and Sec61p (Figure 5B); very little Dsl1p was found in the supernatant with the cytosolic marker protein PGK.

Membrane extractions were performed to test whether Dsl1p is a peripheral membrane protein, as would be expected from the data mentioned above and the lack of an apparent transmembrane domain in the predicted primary sequence of the protein (VanRheenen et al., 2001). Crude yeast membranes were isolated on a buoyant density step gradient and then incubated either with buffer, Triton X-100, 1 M NaCl, or pH 11 sodium carbonate. After centrifugation at 175,000 × g the supernatant (S) and pellet (P) fractions were analyzed by immunoblotting with antibodies against Dsl1p (Figure 5C). Buffer alone did not remove Dsl1p, again indicating that it tightly associates with membrane. On incubation with Triton X-100, Dsl1p is partially released into the supernatant fraction, confirming its membrane association. Dsl1p was not extracted from the membrane by high salt treatment, which is unusual for a peripheral membrane protein and suggests that Dsl1p's interaction with the membrane is not solely through ionic interactions. Last, Dsl1p is efficiently removed upon exposure to pH 11 sodium carbonate. This extraction behavior indicates that Dsl1p is a tightly associated peripheral membrane protein.

Because Dsl1p functions at the ER/Golgi interface, we investigated through subcellular fractionation whether Dsl1p associates with the membranes of either of these organelles. Thus, membranes from a wild-type strain bearing the Golgi resident protein Och1p tagged with an HA-epitope tag (Harris and Waters, 1996) were subjected to sucrose density centrifugation followed by fractionation and immunoblotting with α-Dsl1p, α-Kar2p, and α-HA antibodies (Figure 6A). Dsl1p cofractionated with the ER resident Kar2p, and overlapped only to a limited extent with peak of Och1p-HA. To confirm that the cofractionation of Dsl1p and an ER marker was due to association with the ER rather than another cofractionating organelle we also performed the centrifugation in the presence of EDTA, which causes a reduction in the density of the ER due to dissociation of ribosomes (Antebi and Fink, 1992). We found that the localization of Och1p-HA in the gradient was unchanged, but that both Dsl1p and Kar2p were shifted to less dense fractions (our unpublished results). This fractionation behavior is consistent with Dsl1p residence on the ER membrane.

Confirming the fractionation behavior, double-label immunofluorescence microscopy of Dsl1p-HA revealed a perinuclear staining similar to that of Kar2p (Figure 6B). Together, the cell fractionation and immunofluorescence data strongly suggest that Dsl1p is peripheral membrane protein localized predominantly to the ER at steady state.

Immunoisolation of Dsl1p-interacting Proteins

Because Dsl1p is tightly associated with the ER membrane, we used an affinity approach in an effort to isolate membrane-associated Dsl1p-interacting proteins. First, we screened a number of nonionic detergents and found that Triton X-100H (the hydrogenated form of Triton X-100, which is UV-transparent) and n-dodecyl-β-d-maltoside fairly efficiently (∼50%) extract Dsl1p from microsomal membranes (our unpublished results). Next, to examine whether the extracted Dsl1p associates with other proteins, we subjected the detergent extracts to size exclusion chromatography followed by immunoblotting with α-Dsl1p antibodies (Figure 7A). For both detergents Dsl1p was found to chromatograph at an inordinately large size, comparable with an ∼700–800-kDa globular protein. This size, however, is only an estimate due to the presence of detergent and because the shape of the complex is not known. Nevertheless, this chromatographic behavior suggested that detergent-extracted Dsl1p may stably associate with other proteins.

To isolate Dsl1p interacting proteins, membranes from a dsl1Δ strain bearing DSL1 or DSL1-HA on a low-copy number plasmid were extracted with Triton X-100H, the extracts were clarified by centrifugation to remove aggregated material, and the supernatants were immunoprecipitated with α-HA antibody. Visualization of the proteins in the immunoprecipitates after SDS-PAGE and silver staining revealed that proteins of ∼90-, 80-, and 55-kDa apparent molecular mass were specifically and reproducibly harvested from the Dsl1p-HA strain (Figure 7B). Minor proteins of ∼40 and 50 kDa were also reproducibly obtained. Immunoblotting of the immunoprecipitates with α-Dsl1p and α-HA antibodies showed that the 90-kDa protein was Dsl1p-HA, and the minor 40- and 50-kDa polypeptides were proteolytic fragments of Dsl1p-HA (our unpublished results). The 80- and 55-kDa proteins were not recognized by α-Dsl1p or α-HA antibodies and therefore represent Dsl1p-interacting proteins.

As shown above (Figure 4), DSL1 interacts genetically with both SEC20 and TIP20. These genes encode proteins with apparent molecular masses on SDS-PAGE of ∼50 and 80 kDa, respectively (Sweet and Pelham, 1992, 1993), very similar to those of the Dsl1p-interacting proteins (Figure 7B). In addition, Tip20p interacts with Sec20p, and both proteins are localized to the ER (Sweet and Pelham, 1993) and function in retrograde traffic (Cosson et al., 1997). Additional evidence suggesting that the Dsl1p-interacting proteins might be Sec20p and Tip20p has been provided by a recent genome-wide two-hybrid screen of yeast protein–protein interactions (Ito et al., 2001), which revealed interactions between Dsl1p and Tip20p. The interaction seems reliable because an interaction was observed irrespective of whether the proteins are fused to the DNA-binding or activation domains, and because each protein has few other partners.

To address whether the 80-kDa Dsl1p-interacting protein is Tip20p, we sought to coimmunoprecipiate Dsl1p from a strain bearing Tip20p with a C-terminal triple-myc epitope tag (Figure 7C). Indeed we found that Dsl1p associates with Tip20p-myc and that the immunopreciptiation could be blocked by inclusion of competitor myc peptide, showing that the immunoprecipitation is specific (Figure 7C, lane 4). As another control, we probed the immunoprecipitate for the presence of Sec61p, but were unable to detect this ER-resident protein (Figure 7C, bottom), again suggesting that the Dsl1p-Tip20p interaction is specific. The immunoprecipitation was also quite efficient, with ∼30–40% of the Dsl1p coimmunoprecipitating with the Tip20p-myc.

Two-Hybrid Interactions of DSL1 with Retrograde Transport Components

In a parallel effort to identify proteins that physically interact with Dsl1p we performed a yeast two-hybrid screen with Dsl1p fused to the Gal4p-DNA binding domain as bait. The strain used in this screen tends to have fewer false positives than previous designs because it uses three Gal4p-driven genes with different promoter regions (James et al., 1996). We isolated three Dsl1p-interacting components in the screen, each one multiple times. Two were represented by uncharacterized open reading frames and the third was the Ret2p/δ-COP component of the retrograde COPI coat (Figure 8A). We obtained three distinct but overlapping fragments of Ret2p/δ-COP (Figure 8B); the region common to all the isolates encodes approximately the central one-half of the protein. This finding, in conjunction with our previous demonstration of genetic interactions between DSL1 and genes encoding subunits of the COPI coat (VanRheenen et al., 2001), suggests that Dsl1p physically interacts with Ret2p/δ-COP and raises interesting possibilities about the molecular function of Dsl1p in retrograde traffic.