Figure 4. Ser57 phosphomimetic mutations in ubiquitin accelerate endocytic trafficking.
(A) Analysis of yeast growth in the presence of canavanine. In this experiment, the indicated ubiquitin variants (wildtype, Ser57Ala, or Ser57Asp) were expressed exogenously from the pRS416 plasmid under the control of the ADH1 promoter in the SEY6210 strain background. Canavanine hypersensitivity is indicative of an endocytic defect, while canavanine resistance is indicative of hyper-active endocytic trafficking. (B) Analysis of yeast growth in the presence of canavanine. Here, the indicated ubiquitin variants (wildtype, Ser57Ala, or Ser57Asp) were expressed as the sole source of ubiquitin from the native RPS31 promoter in the SUB280 strain background. (C) Flow cytometry analysis of yeast cells expressing Mup1-pHluorin grown in the absence of methionine and then stimulated by addition of a low concentration of methionine (667ng/mL). Fluorescence of pHluorin is pH-sensitive and lost during endocytic trafficking when the cargo encounters an acidic environment. Triplicate experiments are shown for cells expressing wildtype (circles) or S57D phosphomimetic (squares) ubiquitin. (D) Data generated in (C) were averaged (n = 3, error bars indicate standard deviation) and linear regression was used to generate trend lines and estimate the half-life of Mup1-pHluorin in the context of wildtype or Ser57Asp ubiquitin, as indicated in the table (right).
Figure 4—figure supplement 1. Hyper-active endocytic trafficking in the presence of Ser57Asp phosphomimetic ubiquitin requires conjugation.
Figure 4—figure supplement 2. Hyper-active endocytic trafficking in the presence of Ser57Asp phosphomimetic ubiquitin requires K63-linked poly-ubiquitination.
Figure 4—figure supplement 3. Expression of Ser57Asp phosphomimetic ubiquitin accelerates methionine-stimulated endocytic trafficking of Mup1.
