5 B)

5 B). Rom2p K-Ras G12C-IN-1 in vesicles. Our results indicated that Rho1p is kept inactive in secretory organelles and is activated on its arrival at the plasma membrane, where Rom2p is localized. Results GS is transported to the plasma membrane through the secretory pathway We analyzed the biosynthetic and transport processes of nascent GS after synthesis of the subunit proteins Rho1p and Fks1p/2p. To examine how Rho1p and Fks1p/2p are transported to the plasma membrane, we observed their localization when vesicular transport was blocked by mutations (Kaiser et al., 1997). Consistent with previous reports (Yamochi et al., 1994; Qadota et al., 1996; Ayscough et al., 1999), immunofluorescent microscopic observations revealed that Rho1p and Fks1p/2p were localized at the site of growth in HNPCC1 wild-type cells incubated at 25C or shifted to 37C and incubated for 2 h (Fig. 1 and unpublished data). Rho1p and Fks1p/2p were also localized at the site of growth in mutant cells incubated at 25C (unpublished data). The localization of Rho1p and Fks1p/2p in mutant cells did not alter by a shift to 37C and a subsequent incubation for 10 min (unpublished data). However, after incubation of mutant cells at the restrictive temperature for 2 h, Rho1p and Fks1p/2p were detected not at the site of growth, but in intracellular organelles (Fig. 1 and unpublished data). In and cells, both of which are defective in transport from the ER to the Golgi, Rho1p and Fks1p/2p were mislocalized to the cytoplasm and had a punctate appearance. In and cells with defects in transport from secretory vesicles to the plasma membrane, Rho1p and Fks1p/2p were ubiquitously present. Introduction of the additional mutation of mutant cells (Fig. 1). These results implied that Rho1p and Fks1p/2p localized in mutant cells before the temperature shift were degraded, and that the intracellular proteins observed after the temperature shift were newly synthesized proteins in the exocytic pathway. On the basis of these results, Rho1p and Fks1p/2p may well be transported to the plasma membrane through the secretory pathway after their synthesis on the K-Ras G12C-IN-1 ER. Open in a separate window Figure 1. Localization of Rho1p and Fks1p/2p in cells shifted to 37C. Cells were cultured in YPD at 25C, shifted to 37C and cultured for 2 h. Cultured cells were fixed with formaldehyde and then stained for immunofluorescence microscopy with the anti-Rho1p antibody (left) or the anti-Fks1p/2p antibody (right). Strains used were as follows: wild-type (YPH500), cells cultured at the restrictive temperature for 2 h after growth at the permissive temperature and were used to examine whether Rho1p and Fks1p/2p are detected in secretory vesicle fractions. As described previously (Walworth and Novick, 1987; McCaffrey et al., 1991), cell lysate was subjected to differential centrifugations, and the high-speed pellet obtained was fractionated further on the basis of vesicular size by gel exclusion chromatography. First, we examined the distribution of marker enzymes in the final K-Ras G12C-IN-1 fractions. Invertase, a marker enzyme of secretory vesicles, was eluted from the column as a single peak with its maximum at fraction 23 (Fig. 2 A, right). Plasma membrane ATPase accumulated in secretory vesicles by mutation was co-eluted with invertase. Next, we examined the distribution of Rho1p and Fks1p/2p by immunoblotting analysis and found that the distribution of Fks1p/2p was indistinguishable from in the elution profile of invertase (Fig. 2 B, right). In cells, Rho1p was also found in the secretory vesicle fractions (Fig. 2 B, right), consistent with a preceding report (McCaffrey et al., 1991). By contrast, Rho1p and Fks1p/2p were not distributed to the secretory vesicle fractions in wild-type cells, but were detected in fractions centering at 15 (Fig. 2 B, left), which coincided with the those of plasma membrane based on plasma membrane ATPase activity measurements (Fig. 2 A, left). Thus, Rho1p and Fks1p/2p are indeed localized in secretory vesicles when vesicular transport is blocked by the mutation. Open in a separate window Open in a separate window Open in a separate window Figure 2. Secretory vesicle fractions of (right) cells.