Ear1p interacts with Rsp5p, and it is homologous to Ssh4p. (A) Immunoprecipitation of Ear1p-3HA (wild-type or PPxY mutants) expressed in wild-type cells by using an anti-HA antibody. Input fractions (INP), unbound material (UNB), and IPs were immunoblotted with the indicated antibodies. (B) Schematic primary structure of Ear1p and Ssh4p. TMD, transmembrane domain; B30.2/SPRY, B30.2/SPRY domain; PY, PPxY motifs.

Ear1p/Ssh4p are involved in cargo ubiquitylation at MVBs. (A) Subcellular localization of Ub-GFP-Phm5p wild-type and ear1Δ ssh4Δ cells. Bar, 5 μm. (B) Lysates of wild-type, ear1Δ ssh4Δ, and rsp5 cells expressing Sit1p-GFP were subjected to immunoprecipitation in denaturing conditions with a monoclonal GFP antibody, and then they were immunoblotted with a polyclonal anti-GFP or a monoclonal anti-ubiquitin antibody. Arrowhead, Sit1p-GFP; asterisk, GFP-containing degradation products. (C) Subcellular localization of the PPxY-motif containing proteins Sna3p-GFP and GFP-Bsd2p in wild-type and ear1Δ ssh4Δ cells. Bar, 5 μm.

Ear1p is an endosomal protein that is ubiquitylated and targeted to the vacuole. (A) Colocalization of Ear1p-GFP with endosomal markers (Hse1p-mCherry, top and middle; or Vps27p-mCherry, bottom). Ear1p-GFP expression was driven by its own promoter, either at the endogenous locus (top) or on a multicopy plasmid (middle and bottom). Bar, 5 μm. (B) Localization of Ear1p-GFP (multicopy plasmid) in the indicated strains. Bar, 5 μm. (C) Extracts from the indicated strains expressing Ear1p-GFP (multicopy plasmid) were immunoblotted with anti-GFP and anti-PGK antibodies. Arrowhead, Ear1p-GFP; asterisk, GFP-containing proteolytic fragment. GFP panels are from the same blot, but they represent different areas and exposures. (D) Crude extracts were prepared from the indicated strains expressing Ear1p-mCherry with or without His6-tagged ubiquitin, and they were immunoblotted with anti-DsRed antibody. Empty arrowhead, size of ubiquitin-conjugated Ear1p-mCh; solid arrowheads, size of His6-ubiquitin–conjugated Ear1p-mCh. (E) Crude extracts were prepared from ear1Δssh4Δ expressing wild-type (WT) or PPxY mutants of Ear1p-mCh, and then they were immunoblotted with anti-Ds-Red antibody. Empty arrowhead, size of ubiquitin-conjugated Ear1p-mCh. (F) Crude extracts were prepared from wild-type cells expressing Ear1p-3HA with or without His6-tagged ubiquitin, in denaturing conditions. These extracts (INP) were incubated with Ni-NTA Sepharose beads, and His6-tagged (ubiquitylated) proteins retained on the beads were then eluted with SDS sample buffer (El.). Empty arrow head, size of ubiquitin-conjugated Ear1p-HA; solid arrowheads, size of His6-ubiquitin-conjugated Ear1p-HA.

Ear1p and Ssh4p are required for MVB sorting of specific VPS cargoes. (A) Localization of cargo GFP-fusion proteins in wild-type and ear1Δ ssh4Δ cells. Bar, 5 μm. (B) Crude extracts were prepared from wild-type and ear1Δ ssh4Δ cells expressing GFP-Gap1p, after 120 min of galactose induction and the indicated times of chase. Samples were immunoblotted with anti-GFP or anti-PGK (loading control) antibodies. Arrowhead, GFP-Gap1p; asterisk, GFP-containing degradation product. (C) Localization of GFP-Smf1p in the indicated strains. Bar, 5 μm. (D) Crude extracts were prepared from the indicated strains expressing GFP-Smf1p and immunoblotted with the indicated antibodies. Arrowhead, GFP-Smf1p; asterisk, GFP-containing degradation product.

Ear1p/Ssh4p are required for MVB sorting of the endocytic cargo Fur4p. Fur4p-GFP expression was induced in wild-type and ear1Δ ssh4Δ cells, and chased before triggering its endocytosis by addition of cycloheximide. (A) Intracellular localization of Fur4p-GFP at the indicated time after endocytosis. Arrowheads indicate vacuolar membrane labeling. Bar, 5 μm. (B) Crude extracts were prepared from cells treated as described in A, and then they were immunoblotted with an anti-GFP or an anti-PGK antibody. For an unknown reason, the amount of Fur4p-GFP synthesized in the double-mutant was lower than in the wild type.

Ear1p overexpression affects Rsp5p localization and function toward an Ear1p-independent cargo. (A) Subcellular localization of Sit1p-GFP in Ear1p-mCherry–overexpressing cells after induction and the indicated time of chase. Bar, 5 μm. (B) Wild-type cells, rsp5 mutant cells, and wild-type cells overexpressing Ear1p-mCherry (WT or PPxY mutants) were serially diluted and spotted on SC solid medium supplemented or not with cadmium, and grown for 3 d at 30°C. (C) Localization of GFP-Rsp5p and GFP-Rsp5p(ΔC2) was addressed in wild-type cells or in cells overexpressing Ear1p-mCherry. Arrowheads indicate plasma membrane labeling. The asterisk denotes a cell in which Ear1p-mCherry is not expressed, leading to a wild-type localization of GFP-Rsp5p. For additional pictures, see Supplemental Figure S9. Bar, 5 μm.