Abstract
Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM–endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.
Highlights
In order to adapt to environmental cues, cells must constantly regulate the protein and lipid composition of their plasma membrane (PM); one such mechanism to ensure this is endocytosis
We found that Art2 is the main regulator of substrate- and stressinduced ubiquitylation and endocytosis of the thiamine transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72
We searched for proteins that underwent CHX-induced endocytosis in a WT strain but that remained stabilized at the PM in an artΔ strain (Tables 1 and 2)
Summary
In order to adapt to environmental cues, cells must constantly regulate the protein and lipid composition of their plasma membrane (PM); one such mechanism to ensure this is endocytosis. The endocytosis of many yeast transporters is triggered by the addition of an excess of their substrate or ligand [1,2,3]. In yeast, this process is triggered by protein ubiquitylation, catalyzed by a single ubiquitin ligase Rsp. Similar to the human ortholog Nedd, Rsp is a family member of the homologous to E6 associated protein carboxyl terminus (HECT)-type ubiquitin ligases. The common feature between all Rsp targets is the presence of an [L/V/P]PxY motif, which interacts with the WW domains of Rsp5 [4].
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