Figure 5.

Npl4-Cdc48 implicated in Ist1-mediated recycling pathway. (A) Schematic representation of model for how yeast Ist1 could drive recycling, based on studies on human IST1 that drives polymerization/scission of endosome tubules to return material to the surface. (B) Efflux measurements were recorded from ist1∆ cells transformed with either vector control or plasmids expressing Ist1^WT or Ist1^∆MIM loaded with FM4-64 for 8 min at RT followed by ice-cold washes. Cartoon representation of domain interaction between Vps4 and Ist1 included above. (C) Airyscan confocal microscopy of Cos5-GFP from a plasmid (left) or stably integrated Ste3-GFP-DUb (right) expressed in WT and vps4∆ cells, and also in the presence of Vps4^EQ expressed from the CUP1 promoter in the presence of 100 µM copper chloride. (D) Stably integrated Ste3-GFP-DUb expressed in WT, ist1∆, and npl4∆ cells imaged by Airyscan confocal microscopy. (E) FM4-64 efflux measurements from indicated strains WT, npl4∆, and ist1∆ cells grown to mid-log phase prior to loading with dye for 8 min at RT and efflux measured after washes. (F) Stably integrated Ste3-GFP-DUb was expressed in strains haboring temperature-sensitive alleles of CDC48 (cdc48-2 and cdc48-3), grown to mid-log phase at 25°C, and imaged by Airyscan confocal microscopy directly or following a 30-min incubation at 30°C. (G–I) Efflux measurements from indicated cells were first loaded with FM4-64 for 8 min, washed three times prior to cytometry. Scale bar, 5 μm.