Internal Amino Acids Promote Gap1 Permease Ubiquitylation via TORC1/Npr1/14-3-3-Dependent Control of the Bul Arrestin-Like Adaptors

Abstract

Ubiquitylation of many plasma membrane proteins promotes their endocytosis followed by degradation in the lysosome. The yeast general amino acid permease, Gap1, is ubiquitylated and downregulated when a good nitrogen source like ammonium is provided to cells growing on a poor nitrogen source. This ubiquitylation requires the Rsp5 ubiquitin ligase and the redundant arrestin-like Bul1 and Bul2 adaptors. Previous studies have shown that Gap1 ubiquitylation involves the TORC1 kinase complex, which inhibits the Sit4 phosphatase. This causes inactivation of the protein kinase Npr1, which protects Gap1 against ubiquitylation. However, the mechanisms inducing Gap1 ubiquitylation after Npr1 inactivation remain unknown. We here show that on a poor nitrogen source, the Bul adaptors are phosphorylated in an Npr1-dependent manner and bound to 14-3-3 proteins that protect Gap1 against downregulation. After ammonium is added and converted to amino acids, the Bul proteins are dephosphorylated, dissociate from the 14-3-3 proteins, and undergo ubiquitylation. Furthermore, dephosphorylation of Bul requires the Sit4 phosphatase, which is essential to Gap1 downregulation. The data support the emerging concept that permease ubiquitylation results from activation of the arrestin-like adaptors of the Rsp5 ubiquitin ligase, this coinciding with their dephosphorylation, dissociation from the inhibitory 14-3-3 proteins, and ubiquitylation.

View publisher note:

The Running of the Buls: Control of Permease Trafficking by α-Arrestins Bul1 and Bul2

ACKNOWLEDGMENTS

We thank S. Léon and R. Haguenauer-Tsapis for regular and fruitful discussions, M. Hall for the pAS103 plasmid, Pascual Sanz for the pGST and pGST-Bmh2 plasmids, and Gerry Fink for the bmh strain. We also thank Catherine Jauniaux for her invaluable contribution to isolating strains and plasmids and Lydia Spedale for her technical assistance.

This work was supported by an FRSM grant (3.4.592.08.F) and an ARC grant (AUWB 2010-15-2) of the Fédération Wallonie-Bruxelles and by the CIBLES grant (grant 716760) of the Région Wallonne de Belgique.