Figure 3.

Sorting of CPY and endocytic cargoes upon Vps8 overproduction. (A) Sorting of an endocytosed receptor. Localization of Ste3-GFP was determined in wild-type, vps3Δ, vps8Δ, and Vps8– and Vps21–co-overexpressing (GAL1-VPS8, ADH1-VPS21) cells by fluorescence microscopy after vacuole staining with FM4-64 and cycloheximide treatment. Ste3-GFP, which is sorted into the vacuole lumen in wild-type cells, is mislocalized in vps3Δ and in vps8Δ cells, consistent with a role of both proteins along the endocytic pathway. Size bar, 10 μm. (B and C) Time-course internalization of FM4-64. Wild-type (B) and Vps8– and Vps21–co-overexpressing (GAL1-VPS8, ADH1-GFP-VPS21; C) cells were incubated for 2 min at 30°C with FM4-64, chased for the indicated times, and analyzed by fluorescence microscopy as in Figure 1A.