Stress Conditions Promote Yeast Gap1 Permease Ubiquitylation and Down-regulation via the Arrestin-like Bul and Aly Proteins

Myriam Crapeau,Ahmad Merhi,Bruno André

doi:10.1074/jbc.m114.582320

Myriam Crapeau, Ahmad Merhi + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m114.582320

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Abstract

Gap1, the yeast general amino acid permease, is a convenient model for studying how the intracellular traffic of membrane transporters is regulated. Present at the plasma membrane under poor nitrogen supply conditions, it undergoes ubiquitylation, endocytosis, and degradation upon activation of the TORC1 kinase complex in response to an increase in internal amino acids. This down-regulation is stimulated by TORC1-dependent phosphoinhibition of the Npr1 kinase, resulting in activation by dephosphorylation of the arrestin-like Bul1 and Bul2 adaptors recruiting the Rsp5 ubiquitin ligase to Gap1. We report here that Gap1 is also down-regulated when cells are treated with the TORC1 inhibitor rapamycin or subjected to various stresses and that a lack of the Tco89 subunit of TORC1 causes constitutive Gap1 down-regulation. Both the Bul1 and Bul2 and the Aly1 and Aly2 arrestin-like adaptors of Rsp5 promote this down-regulation without undergoing dephosphorylation. Furthermore, they act via the C-terminal regions of Gap1 not involved in ubiquitylation in response to internal amino acids, whereas a Gap1 mutant altered in the N-terminal tail and resistant to ubiquitylation by internal amino acids is efficiently down-regulated under stress via the Bul and Aly adaptors. Although the Bul proteins mediate Gap1 ubiquitylation of two possible lysines, Lys-9 and Lys-16, the Aly proteins promote ubiquitylation of the Lys-16 residue only. This stress-induced pathway of Gap1 down-regulation targets other permeases as well, and it likely allows cells facing adverse conditions to retrieve amino acids from permease degradation.

Highlights

Gap1 is a yeast amino acid permease ubiquitylated in response to internal amino acids
As previous reports describe TORC1 inhibition in response to stress [31,32,33], it seems likely that the stress conditions tested here cause Gap1 down-regulation by inhibiting TORC1
The Aly1 and Aly2 proteins were found to control Gap1 trafficking, but in a manner opposite to what we observe under stress conditions [28]; the authors found Gap1 to be less abundant at the plasma membrane of aly1⌬ aly2⌬ cells, suggesting a role of Aly proteins in Gap1 recycling from endosomes to the cell surface

Summary

Background

Gap is a yeast amino acid permease ubiquitylated in response to internal amino acids. Present at the plasma membrane under poor nitrogen supply conditions, it undergoes ubiquitylation, endocytosis, and degradation upon activation of the TORC1 kinase complex in response to an increase in internal amino acids This down-regulation is stimulated by TORC1-dependent phosphoinhibition of the Npr kinase, resulting in activation by dephosphorylation of the arrestin-like Bul and Bul adaptors recruiting the Rsp ubiquitin ligase to Gap. The Bul proteins mediate Gap ubiquitylation of two possible lysines, Lys-9 and Lys-16, the Aly proteins promote ubiquitylation of the Lys-16 residue only This stress-induced pathway of Gap down-regulation targets other permeases as well, and it likely allows cells facing adverse conditions to retrieve amino acids from permease degradation. Recent work has shown that the ubiquitylation of specific permeases can be triggered by direct reg-

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION