fig8: The Ypt32p binding site on Sec2p is located downstream from the Sec4p binding site. (A) In parallel experiments, Ypt32-GTP-γ-S or Sec4p (both 30 ng per tube; final concentration in the binding assay 3 nM) were incubated with full-length GST–Sec2p or various GST–Sec2 truncated proteins (Fig. 7) immobilized on beads. The beads were then washed and the bound proteins were resolved by SDS-PAGE and detected by Western blot analysis. Input represents 2 and 4% of total Ypt32p and 10 and 20% of total Sec4p protein used per binding mixture. (B) The NH2-terminal domain (aa 1–160) of Sec2p has full exchange activity on Sec4. [3H]GDP release from Sec4p (0.2 μM) in the total reaction volume of 60 μl was measured. 20 μl of beads containing attached GST–Sec2 constructs (for purification procedure see Materials and methods section) were included in the reaction mixtures. At the times indicated, 10 μl of reaction mixtures was withdrawn and Rab-bound radioactivity was determined by the filter-binding assay. Data represent one of two independent similar experiments.

Mentions: To further define the Ypt32p and Sec4p binding sites on Sec2p, expression vectors for several truncated Sec2p alleles have been constructed and the corresponding proteins were expressed as GST fusions in yeast. As shown in a schematic diagram (Fig. 7 A), the constructs included the truncated proteins encoded by the ts allele sec2-59 (aa 1–374) and the point mutant sec2-78 (Sec2C483Y) as well as additional truncations Sec2(aa 1–160), Sec2(aa 161–759), and allele sec2-70 (aa 1–508). All of the fusion proteins were purified from yeast lysates. Purification yielded bands of the appropriate molecular weights (Fig. 7 B). These constructs were immobilized on glutathione-Sepharose beads and tested for their ability to bind Sec4p and Ypt32p (Fig. 8 A). Binding of Ypt32p to all fusion proteins except GST–Sec2(aa 1–160)p was observed. Based on the differential binding of Ypt32p to GST–Sec2-59(aa 1–374)p and GST–Sec2(aa 1–160)p, we believe that the Ypt32p binding site on Sec2p is located within the NH2-terminal half of the Sec2p molecule, between amino acids 160 and 374. On the other hand, all of the Sec4p binding determinants appear to be located within the NH2-terminal coiled coil domain (aa 1–160) of Sec2p. The domain composed of the first 160 amino acids of Sec2p is also fully sufficient for its exchange activity (Fig. 8 B). No other part of the Sec2p molecule appears to contribute to the ability of Sec2p to catalyze the nucleotide exchange on Sec4p in vitro.

Ortiz D, Medkova M, Walch-Solimena C, Novick P - J. Cell Biol. (2002)

Bottom Line: Temperature-sensitive (ts) mutations in sec2 and sec4 result in a tight block in secretion and the accumulation of secretory vesicles randomly distributed in the cell.Although the ts mutation sec2-78 does not affect nucleotide exchange activity, the protein is mislocalized.Third, Ypt32p and Sec2p interact biochemically, but Sec2p has no exchange activity on Ypt32p.

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.

Abstract: SEC2 is an essential gene required for polarized growth of the yeast Saccharomyces cerevisiae. It encodes a protein of 759 amino acids that functions as a guanine nucleotide exchange factor for the small GTPase Sec4p, a regulator of Golgi to plasma membrane transport. Activation of Sec4p by Sec2p is needed for polarized transport of vesicles to exocytic sites. Temperature-sensitive (ts) mutations in sec2 and sec4 result in a tight block in secretion and the accumulation of secretory vesicles randomly distributed in the cell. The proper localization of Sec2p to secretory vesicles is essential for its function and is largely independent of Sec4p. Although the ts mutation sec2-78 does not affect nucleotide exchange activity, the protein is mislocalized. Here we present evidence that Ypt31/32p, members of Rab family of GTPases, regulate Sec2p function. First, YPT31/YPT32 suppress the sec2-78 mutation. Second, overexpression of Ypt31/32p restores localization of Sec2-78p. Third, Ypt32p and Sec2p interact biochemically, but Sec2p has no exchange activity on Ypt32p. We propose that Ypt32p and Sec4p act as part of a signaling cascade in which Ypt32p recruits Sec2p to secretory vesicles; once on the vesicle, Sec2p activates Sec4p, enabling the polarized transport of vesicles to the plasma membrane.