Interaction between Sec2p and the exocyst. (A) Exocyst subunits are present in GST-Sec2p pull downs from yeast. To induce expression of GST-Sec2p (NY2432), GST-Gea2p (SFNY1311), GST-Vps1p (SFNY 1586), and GST (NY2433), the cells were grown in YP medium containing 2% galactose. GST pull downs were carried out as described in Materials and Methods. The efficiency of GST-Sec2p, GST-Gea2p, and GST-Vps1p pull downs was 40–50%, whereas the efficiency of GST pull down was 90%. Western blots were probed with αSec10p, αSec15p, αSec8p, αTrs33p, and αAdhp. The relative amount of GST-fusion protein in the pull downs assessed by blotting with αGST. The input represents 1% of lysate prepared from yeast strain NY2432. Lysates from all strains used in this experiment were adjusted to the same protein concentration before pull downs. The amounts of Sec8p, Sec10p, Sec15p, Trs33p, and Adhp were equal in these lysates and corresponded to the endogenous amount present in a wild-type strain. (B) Purified GST-Sec2p interacts with purified exocyst in vitro. GST-Sec2p (purified from NY2432) immobilized on glutathione-Sepharose beads (20 μl of 50% slurry of beads, the estimated amount of GST-Sec2p is 0.5 μg) was incubated for 1 h at room temperature with intact exocyst complex purified from NY2520 (lane 1) in a buffer containing 20 mM PIPES, pH 6.8, 150 mM NaCl, 0.5 mg/ml BSA, 1 mM magnesium acetate, 1 mM imidazole, 2 mM CaCl₂, 0.1% Igepal 30, and 10 mM 2-mercaptoethanol in a total volume of 200 μl. The purification of the exocyst is described in Materials and Methods. The control (lane 2) represents exocyst binding to the GST protein immobilized on glutathione-Sepharose beads. The amount of endogenous exocyst copurifying with GST-Sec2p or GST from yeast (lanes 3 and 4) is negligible compared with the amount of purified exocyst added to the binding mixtures. The input represents 5 and 10% of the purified exocyst used in the binding mixtures. The exocyst subunits, Sec10p and Sec15p were detected with αSec10p and αSec15p antibody, respectively. Sec5-HA fusion protein was detected with αHA antibody. The results shown represent one of three independent experiments.

The Sec15p binding site is on the N-terminal half of Sec2p, whereas the “localization domain” (aa.451-508) is inhibitory to binding. (A) Sec2p constructs N-terminally tagged with GST were co-overexpressed with Sec15p (NY2532-2543). Cells were grown overnight in YP + 2% glycerol and induced by adding 2% galactose for 8 h. Fusion proteins were pulled down using glutathione-Sepharose beads as described in Materials and Methods, and the Sec15p present in these pull downs was detected with αSec15p antibody. For a loading control, GST-Sec2p constructs were detected by αGST antibody. (B) Sec2p and Sec2-78p N-terminally tagged with GST were overexpressed in yeast with (NY2532 and NY2540) or without (NY2432 and NY2438) concurrent overexpression of Sec15p. Complex formation between Sec2-78p and Sec15p (lane 2) can be detected by staining SDS-PAGE gels with Coomassie brilliant blue. (C) The binding sites for Sec15p and Ypt32p on Sec2p overlap. GST-Sec2p constructs were purified from yeast strains (NY2432-2439 and NY2522-2524) using glutathione-Sepharose beads. Binding studies with recombinant His₆-Ypt32p and His₆-Sec15p purified from E. coli were performed as described in Materials and Methods. Ypt32p was preloaded with GTPγS. His₆-Sec15p and His₆-Ypt32p were detected with αSec15p and αYpt32p antibodies, respectively. (D) Summary of Sec15p and Ypt32p binding to Sec2p constructs.

Sec15p and Ypt32p compete for binding to Sec2p. (A) GST-Sec2p or GST was immobilized on glutathione-Sepharose beads (20 μl of 50% slurry, the amount of the fusion protein on beads was 2.5 μg) and incubated with 100 nM His₆-Ypt32p preloaded with GTPγS as described previously (Ortiz et al., 2002). Increasing amounts of His₆-Sec15p (top) or for a control His₆-Sec8p (bottom) were included in the binding mixtures. The binding reaction was performed in a buffer containing 1× PBS, 1 mg/ml BSA, and 0.25% Triton X-100 for 1 h at room temperature. (B) Top, GST-Sec2p or GST immobilized on glutathione-Sepharose beads was incubated with 20 nM His₆-Sec15p and increasing amounts of His₆-Ypt32p-GTPγS. Bottom, GDP-loaded Ypt32p binds GST-Sec2p less efficiently and does not displace Sec15p from GST-Sec2p. All proteins in this experiment were purified from E. coli. His₆-Sec15p and His₆-Ypt32p were detected with αSec15p and αYpt32p antibodies, respectively. (C) The relative affinities of Sec2p-Ypt31p and Sec2-Sec15p interactions. GST-Ypt31p, GST-Sec15(aa.577-910)p and His₆-Sec2p were purified from E. coli by using glutathione-Sepharose and Ni-NTA agarose, respectively. Various amounts of His₆-Sec2p were incubated with 20 nM GST-Ypt31p, GST-Sec15(aa.577-910)p, or GST attached to glutathione-Sepharose beads in a total volume 100 μl for 60 min at room temperature. GST-Ypt31p was preloaded with GTPγS, and no binding was observed for the nucleotide free or GDP bound forms of GST-Ypt31p. The protein samples were subjected to SDS-PAGE, and the amount of bound His₆-Sec2p was determined by Western blotting with αSec2p antibody. Bound His₆-Sec2p was plotted against free His₆-Sec2p and the dissociation constant K_d was estimated by fitting the results with a single rectangular hyperbola equation: B = B_maxL/(K_d + L), where B is bound and L is free His₆-Sec2p. Three independent experiments estimated the dissociation constant K_d of GST–Ypt31p–Sec2p interaction to be 0.65 ± 0.1 μM, whereas the K_d of the GST-Sec15(aa.577-910)p–Sec2p interaction was 0.15 ± 0.05 μM.

Velocity and density gradient analysis of Sec2p-exocyst binding. (A) NY2546 (Sec2p, Sec15-13xmycp), NY2547 (Sec2-GFPp, Sec15-13xmycp), and NY2549 (Sec2-78-GFPp, Sec15-13xmycp) lysates were subjected to velocity gradient centrifugation following detergent solubilization as described in Materials and Methods. Fifteen fractions were collected from the top and analyzed for the presence of Sec2p and Sec15p by Western blotting. From fractions 4 to 9, Sec2-GFPp was immunoprecipitated with anti-GFP, and Sec15-13xmycp present in the immunoprecipitations (IPs) was detected by anti-myc. One percent of Sec15p present in fractions 7, 8, and 9 can be coprecipitated with Sec2-GFPp; 5% of Sec15p present in fractions 7, 8, and 9 can be coprecipitated with Sec2-78-GFPp. No Sec15p was detected in the immunoprecipitates from the control strain containing untagged Sec2p. The efficiency of αGFP precipitation was typically 80%. (B) The sedimentation profiles of Sec2p and Sec15-13xmycp in wild-type (closed symbols) and sec2-78 cells (open symbols) in 10–35% glycerol velocity gradients. (C) Precleared lysates of NY2547 (Sec2-GFPp, Sec15-13xmycp) and NY2549 (Sec2-78-GFPp, Sec15–13xmycp), and NY2546 (Sec2p, Sec15-13xmycp) were loaded at the bottom of a tube containing 35% iodixanol and centrifuged as described in Materials and Methods. Seven fractions were collected from the top. The amount of Sec2p, Sec15p, Ssop, and Adhp in each fraction was determined by Western blotting. The left panels show data for a strain expressing Sec2-GFPp (NY2547); the middle panels show data for a strain expressing Sec2-78-GFPp (NY2549). As a negative control, the identical experiment was also performed with a strain expressing untagged Sec2p (NY2546, right). For each fractionation experiment, a portion of each of the seven fractions was subjected to immunoprecipitation with anti-GFP and Sec15p was detected with anti-myc (bottom). The efficiency of αGFP precipitation was typically 70–80%. The input lane in all IP blots (bottom) represents 2.5% of Sec15-13xmycp present in fraction 1 (lane 8) and 2.5% of total Sec15-13xmycp in each lysate before the fractionation (lane 9). No Sec15-13xmycp was detected in a control αGFP precipitation from a strain expressing untagged Sec2p (NY2546). (D) Precleared lysates of NY2547(Sec2-GFPp, Sec15-13xmycp) and NY2546 (Sec2p, Sec15-13xmycp) were loaded on top of 20–40% sorbitol gradient and centrifuged as described in Materials and Methods. Fourteen fractions were collected from the top, and 2% of each fraction was analyzed for the amount of Sec22p, Sncp, Sec2p, Sec15p, Sec4p, Ssop, and Adh1p by Western blotting with an antibody raised against each protein except Sec15p, which was detected with αmyc. Input represents 0.14% of total protein loaded on top of the gradient. The distribution of markers upon fractionation of lysates prepared from strain NY2547 and a control strain NY2546 looked identical; therefore, only data for NY2547 are shown. A portion of each fraction was immunoprecipitated with αGFP, and the amount of Sec15-13mycp in the immunoprecipitates was determined with αmyc. Sec2-GFPp in the immunoprecipitates was detected with αSec2p. The input lane represents 0.05% of total protein loaded on top of the gradient. Control IPs represent identical immunoprecipitation experiments with the control strain NY2546 expressing untagged Sec2p.

Model of Sec2p function. Sec2p is recruited to secretory vesicles by its interaction with Ypt32p. On the vesicle, Sec2p activates Sec4p. Activated Sec4p in turn recruits its effector Sec15p, which displaces Ypt32p from its binding site on Sec2p. Under normal conditions, Sec2p is released from the exocyst after vesicle tethering. Sec2p mutants defective in the amino acid region 451-508 display increased binding to Sec15p and cannot recycle, which results in mislocalization of Sec2p.

The Sec15p subunit of the exocyst complex interacts with GST-Sec2p. (A) Full-length GST-Sec2p was purified from E. coli strain BL21. As a control, GST protein was also purified from E. coli. GST-Sec2p (lanes 1) and GST (lanes 2) immobilized on glutathione-Sepharose beads (20 μl of 50% slurry, the amount of the fusion protein on beads is 2.5 μg) were incubated with the different exocyst subunits (0.5 μg) individually purified from E. coli. The binding reaction was performed in a buffer containing 1× PBS, 1 mg/ml BSA, and 0.25% Triton X-100 for 1 h at room temperature. Lanes 3 represents 10% of the input of each individually purified exocyst subunit. His₆-exocyst subunits were detected with monoclonal αHis (Novagen). (B) Co-overexpression of GST-Sec2p and Sec15p in yeast results in increased coprecipitation of Sec15p with GST-Sec2p. Top left, GST-Sec2p was co-overexpressed with Sec15p in yeast (NY2525) and isolated using glutathione-Sepharose beads. As a control GST-Sec10p (a known Sec15p interacting protein; positive control) and GST (negative control) were also co-overexpressed with Sec15p (NY2526 and NY2527, respectively). Top right, similar GST pull downs, except that the amount of Sec15p is at native level (NY2528, NY2529, and NY2530). In the Sec15p-overexpressing yeast strains, the amount of Sec15p is 20-fold higher than native amount. Middle, this experiment was analogous to the top experiment, except that in this case, Sec6p was co-overexpressed with GST-Sec2p (NY2550), GST-Sec10p (NY2551), and GST (NY 2552). The exposure times for left and right panels are different. Bottom, amount of Sec15p and Sec6p in overproducing and nonoverproducing yeast lysates. Sec8p is shown as a loading control. In all these experiments, cells were grown overnight in YP + 2% glycerol and induced by adding 2% galactose for 8 h. (C) Binding sites for Sec2p and Sec10p on Sec15p are nonoverlapping. GST-Sec2p immobilized on glutathione beads was incubated with Sec10p (lane 1), Sec15p (lane 2), or both (lane3). Control binding to GST is shown in lane 4. All proteins were expressed and purified from bacteria. The binding reaction was performed as in A. Lanes 5 and 6 represent 10% of input of Sec15p and Sec10p, respectively.

Limited proteolysis patterns for Sec2p and Sec2-78p purified from yeast (NY2432 and NY2438, respectively) are different. Trypsin digestion was allowed to proceed for 5 min, aliquots were withdrawn at t = 0 s, 30 s, 60 s, 2.5 min, and 5 min. Proteolytic fragments were detected with αGST (top) and αSec2p (middle) antibodies and by silver staining of an SDS-PAGE gel (bottom). Both antibodies recognize the N-terminal part of GST-Sec2p and show the same pattern. GST-Sec2 truncated proteins purified from yeast were included (middle) for size comparison with the proteolytic fragments.

Sec15p is present in Sec2-GFPp immunoprecipitates from yeast. Top, Sec2-GFP was immunoprecipitated with αGFP (NY2547, lane 2) from both soluble (S100) and microsomal (P100) fractions. As a negative control, a yeast strain expressing untagged Sec2p was used (NY2546, lane 1). The amount of Sec15-13xmycp in the immunoprecipitates and 1% of input lanes was detected with αmyc. The experiment was conducted as described in Materials and Methods. The efficiency of the αGFP IP was 70–80% from S100 and 30–40% from P100. Immunoprecipitation of GFP-tagged Sec2p mutants Sec2-78-GFPp and Sec2-59-GFPp (NY2548 and NY2549, lanes 3 and 4) by using αGFP results in isolation of larger amounts of Sec15-13xmycp compared with Sec2-GFPp (lane 2). Bottom, amount of Sec2-GFPp (lane 2), Sec2-59-GFPp (lane 3), and Sec2-78-GFPp (lane 4) in the immunoprecipitates is consistent with the fact that in wild-type strain Sec2-GFPp is predominantly present in S100 fraction, whereas in mutant strains a portion of Sec2-59-GFPp and Sec2-78-GFPp redistributes from S100 to P100.

Localization of Sec2-GFPp in a strain overproducing Sec15p. Cells (NY2586) were grown at 25°C in YP-2% glycerol and then induced by adding 2% galactose for 15 h before visualization. Cells were examined with a Zeiss Axioplan2 upright fluorescence microscope as described in Materials and Methods.