Nutrient supply dictates cell signaling changes, which in turn regulate membrane protein trafficking. To better exploit nutrients, cells relocalize membrane transporters via selective protein trafficking. Key in this reshuffling are the α-arrestins, selective protein trafficking adaptors conserved from yeast to man. α-Arrestins bind membrane proteins, controlling the ubiquitination and endocytosis of many transporters. To prevent the spurious removal of membrane proteins, α-arrestin-mediated endocytosis is kept in check through phospho-inhibition. This phospho-regulation is complex, with up to 87 phospho-sites on a single α-arrestin and many kinases/phosphatases targeting α-arrestins. To better define the signaling pathways controlling paralogous α-arrestins, Aly1 and Aly2, we screened the kinase and phosphatase deletion (KinDel) library, which is an array of all non-essential kinase and phosphatase yeast deletion strains, for modifiers of Aly-mediated phenotypes. We identified many Aly regulators, but focused our studies on the TORC1 kinase, a master regulator of nutrient signaling across eukaryotes. We found that TORC1 and its signaling effectors, the Sit4 protein phosphatase and Npr1 kinase, regulate the phosphorylation and stability of Alys. When Sit4 is lost, Alys are hyperphosphorylated and destabilized in an Npr1-dependent manner. These findings add new dimensions to our understanding of TORC1 regulation of α-arrestins and have important ramifications for cellular metabolism.

Cells adapt to changes in their environment through a selective relocalization of their proteome. This reorganization is imperative for cell survival and is regulated, in part, by a highly conserved family of protein trafficking adapters called the α‐arrestins. To help us identify specific regulators of the α‐arrestins and to aid in defining new α‐arrestin functions, we generated and utilized the Saccharomyces cerevisiae Ubiquitin Interactome (ScUbI) library, a unique subset of the deletion collection that contains knockout strains for all non‐essential genes annotated as being important for ubiquitination or ubiquitin interaction. We used this library to screen for factors that altered the ability of α‐arrestins to confer resistance to the TORC1‐inhibiting drug, rapamycin, and found that the most enriched functional category from the screen to be the autophagy gene family, or ATG genes. The ATG genes encode the machinery or regulators of the self‐degradative process of autophagy, which is the process whereby cells recycle damaged components or reclaim nutrients during starvation. Defects in autophagy are linked to many human diseases, neurodegenerative diseases, cancer, and most notably aging. We have defined a genetic network that links α‐arrestins to autophagy. To elucidate the functional connections between α‐arrestins to this pathway, we employed live cell imaging and biochemical analyses with GFP‐Atg8 and Pho8∆60, each of which are established readouts of autophagic flux, and demonstrate that this is impaired in the absence of select α‐arrestins. Electron micrographs suggest a defect in autophagosome biogenesis associated with the loss of α‐arrestins, further supported by biochemical and live‐cell imaging experiments. How then does loss of α‐arrestins lead to impaired autophagosome production? We find defects in lipid droplet maintenance in α‐arrestin mutants suggesting the balance of neutral lipids, a key source of membrane during autophagosome biogenesis, is disrupted when α‐arrestins are lost. These data support an exciting novel role for α‐arrestins as regulators of autophagy, expanding their known suite of functions in sensing and responding to nutrient stress.

Cells selectively reorganize their membrane proteome in response to stressors via selective protein trafficking. The α-arrestins, a family of conserved protein trafficking adaptors, bind to select membrane proteins and interact with the ubiquitin ligase Rsp5. The α-arrestins recruit Rsp5 to its membrane protein substrates, permitting their ubiquitination and endocytosis. To identify new α-arrestin functions, we performed a genetic screen to isolate mutants that alter α-arrestin-mediated resistance to rapamycin, a drug that inhibits TORC1. Interestingly, loss of many of the ATG genes, which encode the machinery needed for the self-degradative process of autophagy, disrupted α-arrestins’ ability to promote growth on rapamycin. Herein we define a genetic network linking α-arrestins to autophagy. We show autophagy impairment in the absence of select α-arrestins, with increased autophagosome lifetimes and delayed/reduced delivery of autophagosomes to the vacuole. The α-arrestin mutants that impeded autophagy had vacuole morphology defects and increased vacuolar retention of Atg18, a member of the PROPPIN family that is needed to maintain vacuole shape and facilitate lipid transfer to expanding autophagosomes. Atg18 binds phosphatidylinositol 3 phosphate (PI3P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), and we observed increased PI3P on the vacuole membrane in α-arrestin mutants. The levels of Vps34 and Fab1, the kinases responsible for the generation of PI3P and PI(3,5)P2, respectively, were also elevated at vacuole membranes in cells lacking α-arrestins. We posit that altered phospholipids in the vacuolar membrane form the basis for the Atg18-Atg2 mislocalization and autophagy defect. These data demonstrate a previously unappreciated link between the α-arrestins and autophagy, expanding the functional impact of these trafficking adaptors in responding to nutrient stress.Author SummaryCells survive nutrient starvation by degrading parts of themselves through the process of autophagy. During autophagy, cells make a double membrane, known as an autophagosome (AP), around bits of cytoplasm or organelles. The AP and its engulfed material are delivered to the vacuole, an organelle that helps break down proteins and lipids. These materials can then be used as building blocks to generate the essential components needed for the cell to survive starvation. For cells to undergo efficient autophagy, they need α-arrestins, a group of proteins important for deciding where membrane proteins localize. In cells lacking α-arrestins, the AP forms slowly, likely due to a problem in growing the AP membrane. This results in less material being delivered to the vacuole via APs when cells do not have α-arrestins. This study defines a new role for α-arrestins in promoting AP formation and starvation survival.

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 α‐arrestins, an emergent and powerful class of protein trafficking adaptors. The α‐arrestins are largely cytosolic proteins that are transiently recruited to membranes via binding to membrane protein motifs. α‐Arrestins bring with them a ubiquitin ligase, which stimulates ubiquitination and subsequent endocytosis of membrane proteins. Phosphorylation of α‐arrestins is key to regulating their trafficking function; dephosphorylated α‐arrestins are generally ‘active’ endocytic adaptors. While some kinases and phosphatases for α‐arrestins are known, the degree of α‐arrestins phosphorylation is staggering, with >40 phosphorylation sites identified for a single α‐arrestin, and suggests we still have much to learn about α‐arrestins regulation. We sought to determine what kinases and phosphates regulate paralogous α‐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 α‐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 α‐arrestins. We focused our studies on the Sit4 protein phosphatase, a key regulator downstream of TORC1 signaling able to influence other α‐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 α‐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 α‐arrestin phosphorylation and stability.