Yeast α-arrestin Art2 is the key regulator of ubiquitylation-dependent endocytosis of plasma membrane vitamin B1 transporters
(2019)
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- Authors
- Supervisors
- Morsomme, Pierre
- 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 and the regulation of trafficking functions of α-arrestins remains largely focused on carbon and amino acid metabolism. Our lab performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into a role of Art2 and Art9 α-arrestins in thiamine (vitamin B1) homeostasis. Indeed, in cycloheximide (CHX)-treated conditions, the thiamine transporter Thi7 was 4- and 11-fold more abundant at the PM in art2Δ and art9Δ strains compared to a wild-type strain, respectively. Fluorescence microscopy and western blot analyses confirmed that Art2 is required for thiamine- and CHX-induced, Rsp5-dependent, endocytosis of the three thiamine transporters Thi7, Nrt1 and Thi72. In this study, we demonstrated that endocytosis of Thi7 requires Art2-dependent ubiquitylation of its cytosolic C-terminal tail. To address the underlying mechanism of Thi7 endocytosis in response to thiamine transport, we developed a genetic screening to isolate transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Two mutants showed strong transport defect: Thi7(N350K) and Thi7(M399R). Then, we generated three-dimensional models of native and mutated Thi7 and simulated thiamine docking to predict the thiamine binding site. Eight mutants appeared to result from mutations of residues forming/surrounding the thiamine binding pocket: Thi7(G59R), Thi7(D85G), Thi7(N133K), Thi7(M247I), Thi7(P286Q), Thi7(N350K), Thi7(V398F) and Thi7(M399R). Therefore, their observed transport defects might be explained by disturbances localized in the binding pocket that prevent interaction and stabilization of thiamine. Then, co-expression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity to accumulate intracellular substrate are important to induce endocytosis. We also provided evidence that Art2-mediated Thi7 endocytosis requires the Sit4 phosphatase, involves the TORC1 complex but is not inhibited by the Npr1 kinase. Additionally, we started to characterize endocytosis of the vitamin B6 transporter Tpn1 and demonstrated that several redundant α-arrestins are required for substrate-induced, Rsp5-dependent, endocytosis and vacuolar targeting of Tpn1.
- Affiliations
Citations
Nootens, S. (2019). Yeast α-arrestin Art2 is the key regulator of ubiquitylation-dependent endocytosis of plasma membrane vitamin B1 transporters. https://hdl.handle.net/2078.5/123996