TY - JOUR
T1 - Yeast α-arrestin Art2 is the key regulator of ubiquitylation-dependent endocytosis of plasma membrane vitamin B1 transporters
AU - Savocco, Jérôme
AU - Nootens, Sylvain
AU - Afokpa, Wilhelmine
AU - Bausart, Mathilde
AU - Chen, Xiaoqian
AU - Villers, Jennifer
AU - Renard, Henri François
AU - Prévost, Martine
AU - Wattiez, Ruddy
AU - Morsomme, Pierre
N1 - Funding Information:
This work was funded by grants from the Fonds de la Recherche Scientifique FRS-FNRS (FNRS-FRFC: 2.4506.12, FNRS-PDR: T.0206.16) to PM and Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture (FRIA) to SN (FNRS-FRIA: 1.E047.16), JS (FNRS-FRIA: 1. E089.14), and JV (FNRS-FRIA: 1.E147.13). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors thank Hugh Pelham, Jürgen Stolz, Bruno André, Sébastien Léon, and Michel Ghislain for providing strains, plasmids, and antibodies. We thank Anna-Maria Marini, Jürgen Stolz, Marc Boutry, and Bernard Hallet for constructive discussions and advice, the SMCS platform of UCLouvain for statistical analysis, and Melissa Page for critical reading.
Publisher Copyright:
© 2019 Savocco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2019/10/1
Y1 - 2019/10/1
N2 - 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.
AB - 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.
KW - Arrestins/genetics
KW - Cell Membrane/drug effects
KW - Cell Wall/drug effects
KW - Endocytosis/genetics
KW - Endoplasmic Reticulum/drug effects
KW - Endosomal Sorting Complexes Required for Transport/genetics
KW - Gene Expression Regulation, Fungal
KW - Membrane Transport Proteins/genetics
KW - Models, Molecular
KW - Mutation
KW - Nucleoside Transport Proteins/genetics
KW - Protein Binding
KW - Protein Kinases/genetics
KW - Protein Phosphatase 2/genetics
KW - Protein Processing, Post-Translational
KW - Protein Structure, Secondary
KW - Proteomics/methods
KW - Saccharomyces cerevisiae/drug effects
KW - Saccharomyces cerevisiae Proteins/genetics
KW - Signal Transduction
KW - Thiamine/metabolism
KW - Transcription Factors/genetics
KW - Ubiquitin-Protein Ligase Complexes/genetics
KW - Ubiquitination
UR - http://www.scopus.com/inward/record.url?scp=85074674246&partnerID=8YFLogxK
UR - https://researchportal.unamur.be/en/publications/yeast-arrestin-art2-is-the-key-regulator-of-ubiquitylationdependent-endocytosis-of-plasma-membrane-vitamin-b1-transporters(3e79c2fc-73cd-4e08-b66e-d55914e92671).html
U2 - 10.1371/journal.pbio.3000512
DO - 10.1371/journal.pbio.3000512
M3 - Article
C2 - 31658248
AN - SCOPUS:85074674246
SN - 1544-9173
VL - 17
SP - e3000512
JO - PLoS biology
JF - PLoS biology
IS - 10
M1 - e3000512
ER -