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. 2006 Jun;17(6):2757–2769. doi: 10.1091/mbc.E05-10-0917

Figure 1.

Figure 1.

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.