Csr2 is ubiquitylated at a conserved site in the ART proteins and this is required for its function in endocytosis. (A, top) Schematic of the primary structure of the Csr2 protein showing potential arrestin-N domain (Becuwe et al., 2012a), arrestin-C domain (Pfam accession no. PF02752), LPxY and PPxY motifs, and the lysine residue identified as ubiquitylated by mass spectrometry (K670; see Fig. S4 A). (bottom) Partial alignment of seven ART proteins from S. cerevisiae showing the conservation of a lysine residue at this position. Arrowheads, residues whose combined mutation abolishes Rod1 ubiquitylation (Becuwe et al., 2012b). (B) Total cell lysates from WT cells expressing a plasmid-encoded Csr2-3HA or its mutated variant, Csr2(KR)-3HA (mutation K670R) were prepared at the indicated times and immunoblotted with the indicated antibodies. (C) Localization of Hxt6-GFP in WT cells (carrying an empty plasmid) and csr2-1 mutant cells, transformed with either an empty plasmid, a plasmid carrying a WT Csr2-3HA, or the point mutant Csr2(KR)-3HA in the indicated growth conditions. (D) From the experiment depicted in C, total cell lysates were prepared and immunoblotted (see a representative immunoblot in Fig. S4 B). GFP signals were quantified at various time points and are plotted ± SEM (n = 3 independent experiments); p-values were calculated for the 7-h time point (***, P < 0.001). (E) WT cells expressing both endogenously tagged Hxt6-VC and either a pTEF-driven Csr2-VN or a pTEF-driven Csr2-KR-VN were grown in glucose medium and switched to lactate medium for 30 min. Csr2-KR-VN-expressing cells were labeled with the vacuolar dye CMAC, washed, and mixed with Csr2-VN-expressing cells. Both strains were imaged together by fluorescence microscopy in the green and blue channels. (left) Detailed view of the indicated cells.
