Figure 6.

Role of the class C core proteins in the Vps21 localization to the LE clusters. (A and B) Localization of GFP-Vps21 (A) or GFP-Vps8 (B) in the indicated strains. Cells were analyzed by fluorescence microscopy as described in Figure 1A. Exposure times were identical for each strain. Size bar, 10 μm. The control of the expression levels is shown on the bottom of panel (A). Cell lysates were prepared from the indicated deletion strains expressing GFP-Vps21 and overexpressing Vps8, and proteins analyzed by SDS-PAGE followed by Western blotting. Vac8 is the loading control. (C) Ultrastructural analyses. Strains used for fluorescence microscopy in panel A: wild-type (image A), GAL1-VPS8 GFP-VPS21 (image B), GAL1-VPS8 GFP-VPS21 vps11Δ (image C), and GAL1-VPS8 GFP-VPS21 vps18Δ (image D) were grown to exponential phase and embedded in Spurr′s resin before being sectioned and imaged. The panels B′, C′, and D′ highlight the clusters of vesicles observed in the strains shown in images B, C, and D, respectively. V, vacuole; M, mitochondrion; N, nucleus. Black bar, 500 nm; white bar, 200 nm. (D) Localization of GFP-Vps21 in strains overexpressing Vps8 and Vps3 (top) or the entire CORVET (bottom). Wild-type, Vps8-overexpressing, and Vps8– and Vps3–co-overexpressing cells were analyzed by fluorescence microscopy. In the bottom panel, diploid cells overexpressing Vps8 or the entire CORVET complex are shown. Size bar, 10 μm.