Differential Phosphorylation Provides a Switch to Control How α-Arrestin Rod1 Down-regulates Mating Pheromone Response in Saccharomyces cerevisiae

Abstract

G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate stimulus-dependent activation of cognate heterotrimeric G-proteins, triggering ensuing downstream cellular responses. Tight regulation of GPCR-evoked pathways is required because prolonged stimulation can be detrimental to an organism. Ste2, a GPCR in Saccharomyces cerevisiae that mediates response of MATa haploids to the peptide mating pheromone α-factor, is down-regulated by both constitutive and agonist-induced endocytosis. Efficient agonist-stimulated internalization of Ste2 requires its association with an adaptor protein, the α-arrestin Rod1/Art4, which recruits the HECT-domain ubiquitin ligase Rsp5, allowing for ubiquitinylation of the C-terminal tail of the receptor and its engagement by the clathrin-dependent endocytic machinery. We previously showed that dephosphorylation of Rod1 by calcineurin (phosphoprotein phosphatase 2B) is required for optimal Rod1 function in Ste2 down-regulation. We show here that negative regulation of Rod1 by phosphorylation is mediated by two distinct stress-activated protein kinases, Snf1/AMPK and Ypk1/SGK1, and demonstrate both in vitro and in vivo that this phospho-regulation impedes the ability of Rod1 to promote mating pathway desensitization. These studies also revealed that, in the absence of its phosphorylation, Rod1 can promote adaptation independently of Rsp5-mediated receptor ubiquitinylation, consistent with recent evidence that α-arrestins can contribute to cargo recognition by both clathrin-dependent and clathrin-independent mechanisms. However, in cells lacking a component (formin Bni1) required for clathrin-independent entry, Rod1 derivatives that are largely unphosphorylated and unable to associate with Rsp5 still promote efficient adaptation, indicating a third mechanism by which this α-arrestin promotes desensitization of the pheromone-response pathway.

Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 α-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein

“…Several examples have been reported in the literature, such as the nitrogen-dependent regulation of Ldb19 (Art1) by the TOR-dependent Npr1 protein kinase and the carbon source-dependent regulation of Rod1 (Art4) by the Snf1 kinase (12,16,19). This latter case was shown to be involved in the regulation of the Jen1 lactate transporter, the Ste2 pheromone receptor, and the Hxt1 and Hxt3 hexose permeases (15)(16)(17).…”

The C-terminal region of the yeast monocarboxylate transporter Jen1 acts as a glucose signal–responding degron recognized by the α-arrestin Rod1

“…Glucose-induced inactivation of Jen1 requires the ␣-arrestin, Rod1/Art4. Activity of Rod1 is regulated by the phosphorylation state, which is modulated by Snf1 kinase and type 1 protein phosphatase (Glc7-Reg1) (14,15). In the absence of glucose, Rod1 is phosphorylated by Snf1 kinase, which facilitates binding to the 14-3-3 proteins (Bmh1/2) to inhibit its interaction with Jen1 (14).…”

Yeast transformation efficiency is enhanced by TORC1‐ and eisosome‐dependent signaling

“…Deletion of YPK1 led to the lowest transformation efficiencies out of all mutant strains we tested in this study. However, further work is needed to clarify the exact role of Ypk1 in DNA uptake because Ypk1 also regulates at least one α‐arrestin (Alvaro, Aindow, & Thorner, ), it impinges on actin dynamics (Niles & Powers, ), and it is involved in the heat stress response (Sun et al., ). The hypotheses that eisosomes mark sites of endocytosis and that transformation is facilitated by endocytosis have one point in common: In both cases, these types of endocytosis differ from well‐studied endocytic pathways, such as clathrin‐mediated endocytosis that originates at actin patches (Kawai et al., ; Ziółkowska, Christiano, & Walther, ).…”