Selective protein trafficking controls the repertoire of membrane proteins at the cell surface. This contingent of surface proteins regulates nutrient/metabolite balance and response to extracellular cues. Environmental changes trigger cellular signaling that drives transitions in the plasma membrane proteome. These alterations are achieved in no small part through regulation of the alpha-arrestins, an emergent and powerful class of protein trafficking adaptors. The alpha-arrestins are largely cytosolic proteins that are transiently recruited to membranes via binding to membrane protein motifs. alpha-Arrestins bring with them a ubiquitin ligase, which stimulates ubiquitination and subsequent endocytosis of membrane proteins. Phosphorylation of alpha-arrestins is key to regulating their trafficking function; dephosphorylated alpha-arrestins are generally 'active' endocytic adaptors. While some kinases and phosphatases for alpha-arrestins are known, the degree of alpha-arrestins phosphorylation is staggering, with >40 phosphorylation sites identified for a single alpha-arrestin, and suggests we still have much to learn about alpha-arrestins regulation. We sought to determine what kinases and phosphates regulate paralogous alpha-arrestins Aly1 and Aly2 using a genetic screen in yeast. Using an array of all known non-essential kinase and phosphatase deletions, we determined which of these influenced alpha-arrestin-mediated resistance to rapamycin, an inhibitor of the TORC1 nutrient-sensing kinase. We identified a large cohort of kinases and phosphates that influenced Aly-dependent phenotypes, causing electrophoretic mobility changes and, in many cases, diminishing the abundance of these alpha-arrestins. We focused our studies on the Sit4 protein phosphatase, a key regulator downstream of TORC1 signaling able to influence other alpha-arrestins, identified in our screen. Strikingly, Aly1 and Aly2 were hyperphosphorylated and destabilized in the absence of Sit4, suggesting that excessive phosphorylation may promote degradation of these alpha-arrestins. For Aly2, but not Aly1, degradation in the sit4 cells could be reversed by loss of the vacuolar protease Pep4, indicating that vacuolar degradation predominates for Aly2 under these conditions. Loss of the Npr1 kinase in sit4 cells restored Aly protein abundances and reversed the hyperphosphorylation, demonstrating that this kinase is responsible for the excess Aly phosphorylation in sit4 cells. This is a remarkable finding as typically Npr1 is considered inactive in sit4, however, we suggest that Npr1 is selectively active in the absence of Sit4, able to modify some substrates but not others. We define new features of TORC1 signaling in regulating alpha-arrestin phosphorylation and stability.

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Journal Article

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Jordahl EM, Bowman RW, Davis S, Ozbaki-Yagan N, Chiang A, Hedayati S, Barsouk H, O'Donnell AF

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