Figure 5.

Apl5 transiently colocalizes with HOPS subunits at the vacuole membrane. Apl5 was C-terminally tagged with either GFP or mCherry. (A) Strains containing these Apl5 fusions were analyzed for ALP maturation using differential centrifugation (see Materials and Methods). (B) Strains containing Apl5-mCherry and a GFP-tagged HOPS subunit (Vps16-GFP, Vps33-GFP, GFP-Vps41, and GFP-Vps39) were grown to mid-log phase and analyzed by fluorescent microscopy. Arrows indicate sites of colocalization between Apl5 puncta and HOPS at the vacuole membrane. For the GFP-Vps39 time average (part B, bottom panels), both GFP and mCherry fluorescence were averaged across 10 frames (∼24 s). (C) Wild-type yeast expressing Apl5-GFP were labeled with FM4-64 to stain the vacuole membrane. For the 10-frame average, both GFP and FM4-64 channels were averaged across 10 frames (∼17 s). Bar, 2 μm. See Supplemental Movies 1–3.