NSF is not essential for CUPS biogenesis. (A) Wild-type (WT) and grh1Δ yeast expressing Vps23-mCherry were cultured in growth conditions or starved for 4 h and analyzed by fluorescence microscopy. (B) Wild-type and grh1Δ strains expressing RFP-Atg9 were cultured in growth conditions or starved and visualized by fluorescence microscopy. Starved cells showing RFP-Atg9–specific punctate elements representing the CUPS were counted in wild-type and grh1Δ cells. 59 ± 3.3% of wild-type cells showed a localization of Atg9 to CUPS compared with only 28.5 ± 1.8% upon deletion of GRH1 (statistically significant with P < 0.0001; error bars represent SEM). (C) Grh1-GFP expressing wild-type yeast and deleted for TLG2, YPT6, and SSO1 were cultured in growth or starvation medium and visualized by fluorescence microscopy. (D) Grh1-GFP was expressed in sec12-4 and sec18-1 strains. Yeast cells were grown at permissive temperature in growth medium and starved for 3 h at either the permissive or the nonpermissive temperature and visualized by fluorescence microscopy. Bar, 2 µm.