Ear1p and Ssh4p are new adaptors of the ubiquitin ligase Rsp5p for cargo ubiquitylation and sorting at multivesicular bodies

Abstract

The ubiquitylation of membrane proteins destined for the vacuole/lysosome is essential for their recognition by the endosomal sorting machinery and their internalization into vesicles of multivesicular bodies (MVBs). In yeast, this process requires Rsp5p, an essential ubiquitin ligase of the Nedd4 family. We describe here two redundant proteins, Ear1p and Ssh4p, required for the vacuolar targeting of several cargoes originating from the Golgi or the plasma membrane. Ear1p is an endosomal protein that interacts with Rsp5p through its PPxY motifs, and it is required for the ubiquitylation of selected cargoes before their MVB sorting. In-frame fusion of cargo to ubiquitin overcomes the need for Ear1p/Ssh4p, confirming a role for these proteins in cargo ubiquitylation. Interestingly, Ear1p is itself ubiquitylated by Rsp5p and targeted to the vacuole. Finally, Ear1p overexpression leads to Rsp5p accumulation at endosomes, interfering with some of its functions in trafficking. Therefore, Ear1p/Ssh4p recruit Rsp5p and assist it in its function at MVBs by directing the ubiquitylation of specific cargoes.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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.

Figure 6.

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.