Arrestin-like proteins mediate ubiquitination and endocytosis of the yeast metal transporter Smf1

Abstract

Many plasma membrane proteins in yeast are ubiquitinated and endocytosed, but how they are recognized for modification has remained unknown. Here, we show that the manganese transporter Smf1 is endocytosed when cells are exposed to cadmium ions, that this endocytosis depends on Rsp5-dependent ubiquitination of specific lysines and that it also requires phosphorylation at nearby sites. This phosphorylation is, however, constitutive rather than stress-induced. Efficient ubiquitination requires Ecm21 or Csr2, two members of a family of arrestin-like yeast proteins that contain several PY motifs and bind to Rsp5. Ecm21 also binds to phosphorylated Smf1, providing a link between Rsp5 and its substrate. PY motif-containing arrestin-like proteins are found in many species, including humans, and might have a general role as ubiquitin ligase adaptors.

Figure 1

Cadmium induces Smf1 internalization through the ubiquitination of lysines 33 and 34. (A) Fluorescent images showing the time course of internalization following the addition of cadmium (Cd), for wild-type GFP-Smf1 (WT) or mutants as indicated; all cells are bsd2Δ. Differential interference contrast images of the cells at the last time point are also shown. (B) Immunoblot of GFP-Smf1 and mutants exposed to cadmium for the indicated times. Cells were end3Δ bsd2Δ, or rsp5 where indicated. Smf1 is found on the surface of both types of cell ((A) and data not shown). (C) GFP-Smf1 immunoprecipitated from cadmium-treated cells expressing Myc-tagged ubiquitin was blotted with anti-GFP and with anti-Myc, which shows ubiquitinated forms. Monoubiquitinated Smf1 is not efficiently detected under these conditions. GFP, green fluorescent protein; Ub, ubiquitin.

Figure 2

Arrestins are required for the efficient ubiquitination and endocytosis of Smf1. (A) Fluorescent images are shown of GFP-Smf1 in the indicated strains, exposed to cadmium for the times indicated. The seven arrestins mutant lacked Ecm21, Csr2, Aly1, Aly2, Rod1, Rog3 and Ygr068c. (B) Immunoblots of GFP-Smf1 in the indicated strains. Cd, cadmium; GFP, green fluorescent protein; Ub, ubiquitin.

Figure 3

Recruitment of Rsp5 by Ecm21. (A) Rsp5-mediated in vitro ubiquitination of Ecm21 and mutants lacking one, two or all three of the carboxy-terminal PY elements. (B) Fluorescent images of GFP-Smf1 co-expressed with Ecm21 (upper panels) or the triple PY mutant of Ecm21 (lower panels) in the seven arrestin mutant cells. Cd, cadmium; GFP, green fluorescent protein; WT, wild type.

Figure 4

Crosslinking of Ecm21 to GFP-Smf1. GFP-Smf1 (omitted in the control) and HA-tagged Ecm21 were co-expressed in ecm21Δ bsd2Δ cells. After spheroplasting and treatment with the indicated concentrations of the DSP crosslinker, the cells were lysed and immunoprecipitated with anti-GFP. Immunoblots of the lysate and immunoprecipitates (IP) with the indicated antibodies are shown. DSP, dithiobis(succinimidyl propionate); GFP, green fluorescent protein; HA, haemagglutinin.

Figure 5

Phosphorylation of Smf1 enhances arrestin binding, ubiquitination and endocytosis. (A) Immunoblot of GFP-Smf1 and the phosphorylation mutants before and after treatment with calf intestinal phosphatase. The SA mutant has the six serines at positions 24, 36 and 51–54 changed to alanines; the SD mutant has the same residues changed to aspartic acid. (B) Fluorescent images of the phosphorylation mutants of GFP-Smf1 in bsd2Δ cells exposed to cadmium (Cd) for the indicated times. (C) Immunoblots of GFP-Smf1 and the phosphorylation mutants, expressed in end3Δ bsd2Δ cells that were exposed to cadmium for the indicated times. Bands above the principal one correspond to ubiquitinated forms. (D) Crosslinking of Ecm21 to GFP-Smf1 and the phosphorylation mutants. The experiment was performed as in Fig 4, with 3 mM DSP crosslinker; control samples lacked GFP-Smf1. DSP, dithiobis(succinimidyl propionate); GFP, green fluorescent protein.