Figure 3.

Ubiquitylation of Rpl25 at K74 regulates 60S ribophagy. (A) Indicated strains were transformed with plasmids encoding either Rpl25-GFP (Rpl25) or rpl25 K74,75R-GFP (rpl25KR) before the deletion of a genomic copy of RPL25. (top) Comparable expression levels of Rpl25-GFP or rpl25 K74,75R-GFP in whole cell lysates were confirmed with anti-GFP blotting. Purified 6His-ubiquitin–conjugated forms of Rpl25-GFP or rpl25 K74,75R-GFP (middle) and 6His-ubiquitin expression (bottom) were analyzed as in Fig. 2. Ub, ubiquitin. (B) Wild-type and ubp3Δ cells expressing Rpl25-GFP or rpl25 K74,75R-GFP were starved for the indicated period of time. (C) Degradation of GFP-tagged proteins was analyzed by anti-GFP blot of whole cell extracts, and the ratio between cleaved GFP and full-length protein was quantified for all time points in four independent experiments. a.u., arbitrary units. (D) Localization of GFP-tagged proteins. C, cytoplasmic localization; V, vacuolar accumulation; C + V, localization in both cytoplasm and vacuole (E) Degradation of Rpl3 was analyzed at 24 h upon starvation by Western blotting of whole cell extracts using anti-Rpl3 (gift from V. Albanese, Institut Jacques Monod, Paris, France) or anti-Mex67 (as a control) antibodies. Rpl3 and Mex67 expression was quantified and normalized to the expression level at time 0. Significance of the differences observed for the ribophagy efficiency was evaluated using Student’s t test. *, P = 0.01–0.05; **, P = 0.001–0.01; ***, P < 0.001. White lanes indicate that intervening lanes have been spliced out. The errors bars correspond to standard deviations.