Figure 3.

Sup35 cleavage occurs in a manner dependent on translation and independently of autophagy. A, wild-type (BY4741) and two autophagy-deficient mutant strains (atg3Δ and atg4Δ) were starved in YPA for 12 h and cultured in YPDA. Cells were harvested at the specified time points and analyzed by Western blotting (WB) using anti-Sup35. B, PrB was purified from the culture supernatant of a pep4Δ strain carrying a PRB1-FLAG single copy plasmid. In vitro Sup35 cleavage reactions were performed by incubating anti-FLAG affinity purified PrB with yeast cell lysate from prb1Δ knock-out strain at 30 °C. An aliquot of the reaction mixture was collected at the indicated time points and analyzed by Western blotting using anti-Sup35. C, in vitro Sup35 cleavage reactions were performed as in B except treatment was with 2 mm PMSF or vehicle control. D, in vitro Sup35 cleavage reactions were performed as in B, except that the condition was at acidic pH (pH 5.0) or neutral pH (pH 7.0). E, glucose-starved GT17 [psi⁻ pin⁻] cells were cultured in YPDA containing either 100 μg/ml of CHX or its vehicle control. Cells were harvested at the specified time points and analyzed by Western blotting using anti-Sup35. F, cell extracts prepared from wild-type (OT60) and prb1Δ (yAO66) were subjected to the polysome profile analysis. Top, absorbance at 254 nm. Bottom, Western blot analysis using anti-Sup35. G, wild-type (BY4741) and two deletion strains of actin-cytoskeleton and endosomal/vacuolar pathways (sla1Δ and end3Δ) were treated as in A.