Abstract
Ubiquitination of the yeast Gap1 permease at the plasma membrane triggers its endocytosis followed by targeting to the vacuolar lumen for degradation. We previously identified Bro1 as a protein essential to this down-regulation. In this study, we show that Bro1 is essential neither to ubiquitination nor to the early steps of Gap1 endocytosis. Bro1 rather intervenes at a late step of the multivesicular body (MVB) pathway, after the core components of the endosome-associated ESCRT-III protein complex and before or in conjunction with Doa4, the ubiquitin hydrolase mediating protein deubiquitination prior to their incorporation into MVB vesicles. Bro1 markedly differs from other class E vacuolar protein sorting factors involved in MVB sorting as lack of Bro1 leads to recycling of the internalized permease back to the plasma membrane by passing through the Golgi. This recycling seems to be accompanied by deubiquitination of the permease and unexpectedly requires a normal endosome-to-vacuole transport function.
Highlights
The machineries responsible for endocytosis and degradation of plasma membrane proteins are crucial to normal regulation of cell surface-associated functions such as transport, signaling, and adhesion [1, 2]
Bro1 markedly differs from other class E vacuolar protein sorting factors involved in multivesicular body (MVB) sorting as lack of Bro1 leads to recycling of the internalized permease back to the plasma membrane by passing through the Golgi
In class E vps mutants, membrane proteins normally transiting through the MVB pathway mainly accumulate in an aberrant late endosome referred to as the class E compartment and/or are missorted to the vacuole limiting membrane
Summary
Plasmids, and Media—The Saccharomyces cerevisiae strains used in this study (Table I) are isogenic with ⌺1278b [38]. Cells were transformed as described previously [22]. Cells were grown in minimal buffered medium (pH 6.1) [39] with 3% glucose as the carbon source except where indicated otherwise. Permease Assays—Cells carrying the gap1⌬ mutation were transformed with the YCpGap or YCpGap1K9K16 plasmid. Yeast Cell Extracts and Immunoblotting—Cells bearing the gap1⌬ mutation were transformed with plasmid YCpGap or YCpGap1K9K16. Extract preparation and Gap immunoblotting were performed a minimum of three times for each strain. Fluorescence Microscopy—Cells transformed with the pJOD10 plasmid were grown in minimal buffered medium (pH 6.1) with 3% galactose and 0.3% glucose as carbon sources and 0.1% proline as the nitrogen source. Glucose was added to the final concentration of 3% 2.5 h before starting the experiment in order to stop Gap1-GFP synthesis. Cells were incubated in medium containing 40 M FM4-64 (Molecular Probes, Eugene, OR), shaken for Strain
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