Activity of a ubiquitin ligase adaptor is regulated by disordered insertions in its arrestin domain

Abstract

The protein composition of the plasma membrane is rapidly remodeled in response to changes in nutrient availability or cellular stress. This occurs, in part, through the selective ubiquitylation and endocytosis of plasma membrane proteins, which in the yeast Saccharomyces cerevisiae is mediated by the HECT E3 ubiquitin ligase Rsp5 and arrestin--related trafficking (ART) adaptors. Here, we provide evidence that the ART protein family members are composed of an arrestin fold with interspersed disordered loops. Using Art1 as a model, we show that these loop and tail regions, while not strictly required for function, regulate its activity through two separate mechanisms. Disruption of one loop mediates Art1 substrate specificity. Other loops are subjected to phosphorylation in a manner dependent on the Pho85 cyclins Clg1 and Pho80. Phosphorylation of the loops controls Art1's localization to the plasma membrane, which promotes cargo ubiquitylation and endocytosis, demonstrating a mechanism through which Art1 activity is regulated.

FIGURE 1:

Art1 contains an arrestin domain disrupted by multiple insertions. (A) Serial dilutions of the indicated yeast strains spotted on synthetic media containing canavanine. (B) Immunoblot of the yeast strains in A expressing Mup1-FLAG before and after treatment with 20 µg/ml Met for 60 min. (C) Immunoblot of yeast expressing Art1 or Art1^2RD with a C-terminal HTF (6xHis-TEV-3xFLAG) tag. Art1-HTF was detected with an α-FLAG antibody. (D) Localization of Art1-mNG and Art1^2RD-mNG in minimal media. Scale bar = 2 µm. Right, quantification of Art1 localization at the PM and Golgi; error bars are 95% CI, n > 300 cells for each condition. (E) Top, Art1 schematic. Nonconserved loop regions shown in gray. Conserved regions predicted to form an arrestin fold are colored. Bottom, disorder confidence predicted DISOPRED3. Gray shading indicates predicted disordered regions. (F) Immunoblot of Art1-HTF tail and loop mutants, with and without the K486R mutation, detected with an α-FLAG antibody. (G) Serial dilutions of Art1 tail and loop mutants spotted on synthetic media.

FIGURE 2:

Art1 loop 3 dictates substrate specificity. (A) Serial dilutions of the Art1 tail and loop mutant strains spotted on media containing canavanine. (B ) Left, immunoblot analysis of Can1-GFP endocytosis induced with the indicated concentration of Arg for 60 min. Right, band intensities were quantified and expressed as the mean % Can1-GFP degradation. Individual experiments are represented as open circles. Error bars indicate 95% CI. n = 3. Filled arrow, p < 0.001; outlined arrow, p < 0.01; dashed arrow, p < 0.05 vs. WT at matched treatment. (C) Immunoblot analysis of Mup1-GFP. As in B, except Met was used to induce endocytosis. n = 3.

FIGURE 3:

Localization of Art1 loop and tail mutants. (A) Localization of the indicated Art1-mNG loop and tail mutants in minimal media and after shifting to rich media for 60 min. Scale bar = 2 µm. (B) Quantification of PM localization of the indicated Art1 loop and tail mutants. (C) Quantification of Golgi localization of the indicated Art1 loop and tail mutants. Bars indicate 95% CI, n > 400 cells for each condition. ***, p < 0.001; **, p < 0.01 vs. WT at matched treatment.

FIGURE 4:

Pho85, and the cyclins Pho80 and Clg1, regulate Art1 activity. (A) Serial dilutions of the indicated null mutants spotted on synthetic media containing canavanine. (B) Immunoblot analysis of Art1-HTF expression in the indicated strains, detected with an α-FLAG antibody. (C) Serial dilutions of WT or cells overexpressing CLG1 spotted on synthetic media containing canavanine. (D) Immunoblot analysis of Mup1-GFP endocytosis induced with the indicated concentration of Met for 60 min. Band intensities were quantified and expressed as the mean % Mup1-GFP degradation. Individual experiments are represented as open circles. Error bars indicate 95% CI. n = 3. Filled arrow, p < 0.001; outlined arrow, p < 0.01; dashed arrow, p < 0.05 vs. WT at matched treatment. (E) Fluorescence microscopy of WT or pho80Δ cells expressing Mup1-GFP, with and without inducing endocytosis by treating with 5 µg/ml Met for 60 min. (F) As in D, except analyzing Can1-GFP endocytosis induced with the indicated concentration of Arg for 60 min. n = 3. (G) As in E, except analyzing Can1-GFP localization after inducing endocytosis with 20 µg/ml Arg for 60 min. (H) As in D, except comparing WT yeast to cells overexpressing CLG1. n = 3. (I) As in E, except comparing WT yeast to cells overexpressing CLG1. (J) As in F, except comparing WT yeast to cells overexpressing CLG1. n = 3. (K) As in G, except comparing WT yeast to cells overexpressing CLG1. Scale bar = 2 µm.

FIGURE 5:

Pho80 and Clg1 affect Art1 localization. (A) Localization of Art1-mNG in WT and pho80Δ yeast in minimal media and after shifting to rich media for 60 min. Right, quantification of Art1-mNG PM and Golgi localization; error bars are 95% CI, n > 250 cells for each condition. *, p < 0.05; ***, p < 0.001 vs. WT at matched treatment. (B) As in A, except comparing Art1-mNG in WT and cells overexpressing CLG1. Scale bar = 2 µm.

FIGURE 6:

Phosphorylation on loops and tails regulate Art1. (A) QMAPS depicting the fold change in phosphopeptide abundance of Art1 in (top) WT vs. clg1Δ or (bottom) WT vs. pho80Δ. (B) Schematic of Art1 showing the location of the phosphorylated residues that when mutated affect Art1 function. (C) Serial dilutions of cells expressing the indicated Art1 mutants spotted on synthetic media containing canavanine. (D) Immunoblot analysis of Mup1-GFP endocytosis induced with the indicated concentration of Met for 60 min. Band intensities were quantified and expressed as the mean % full-length Mup1-GFP remaining. Individual experiments are represented as open circles. Error bars indicate 95% CI. n = 3. Filled arrow, p < 0.001; outlined arrow, p < 0.01; dashed arrow, p < 0.05 vs. WT at matched treatment. (E) Fluorescence microscopy of Mup1-GFP, with and without inducing endocytosis by treating with 5 µg/ml Met for 60 min. (F) As in D, except analyzing Can1-GFP endocytosis induced with the indicated concentration of Arg for 60 min. n = 3. (G) As in E, except analyzing Can1-GFP localization after inducing endocytosis with 20 µg/ml Arg for 60 min. Scale bar = 2 µm.

FIGURE 7:

Phosphorylation affects Art1 localization. Localization of WT Art1-mNG, Art1^S92D,T93D-mNG, or art1^{Clg1 sites → D}-mNG in minimal media and after shifting to rich media for 60 min. Scale bar = 2 µm. Bottom, quantification of Art1 PM localization; error bars are 95% CI, n > 250 cells each condition; ***, p < 0.001 vs. WT at matched treatment.

FIGURE 8:

Model of Art1 regulation. (A) Art1 schematic showing the conserved regions predicted to form the arrestin fold (colored), and the loop and tail regions (gray). (B) Cartoon depicting the predicted organization of the conserved arrestin fold and the localization of the loops. (C) Summary of the effects of Art1 loop region deletions. (D) New modes of Art1 regulation. In addition to the previously identified regulation through Npr1 or Ppz1/2, Art1 phosphorylation/dephosphorylation reactions can be mediated by Clg1-Pho85 and Pho80-Pho85 to regulate its activity. Phosphorylation of Art1 loops and the C-tail regulate its function by affecting its PM localization. When phosphorylated on loop 1, the “miniloop,” and the C-tail, Art1 remains in the cytosol or associated with the Golgi, rendering it inactive. Upon dephosphorylation, Art1 can associate with the PM, where it can recognize its substrate cargoes resulting in ubiquitylation and endocytosis.