FIG 3.

FUS toxicity, aggregation, and turnover are regulated by endocytosis in yeast. (A to C) Serial dilution growth assay of the indicated strains expressing an empty vector or transformed with a plasmid expressing GAL1-regulated FUS-YFP. The strains for which the results are shown in panels A and B are impaired in endocytosis, while the strains for which the results are shown in panel C are impaired in autophagy. (D) Level of FUS-YFP in the indicated strains. The plasmid expressing Vps38-mRuby2 was used for rescue. (E) FUS-YFP focus formation in the indicated strains, based on 3 biological replicates. *, P < 0.05 by analysis of variance with Dunnett’s post hoc test. (F) WT cells with plasmids expressing the empty vector, the vector plus FUS-YFP, Vps34-mRuby2 and FUS-YFP, and Vps38-mRuby2 and FUS-YFP were tested by a growth assay. (G) FUS-YFP protein stability assays following GAL1 transcriptional shutoff (glucose addition). FUS-YFP levels were quantified following normalization to the level for the GAPDH loading control, with the normalized amounts being indicated beneath the gels. (H) Representative Western blot of steady-state levels of FUS-YFP in the indicated strains.