Background information: Phosphatidylinositol (PI) is an essential phospholipid, critical to membrane bilayers. The complete deacylation of PI by B-type phospholipases produces intracellular and extracellular glycerophosphoinositol (GPI). Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters at the yeast plasma membrane. Internalized GPI is degraded to produce inositol, phosphate and glycerol, thereby contributing to these pools. GIT1 gene expression is controlled by nutrient balance, with phosphate or inositol starvation increasing GIT1 expression to stimulate GPI uptake. However, less is known about control of Git1 protein levels or localization. Results: We find that the α-arrestins, an important class of protein trafficking adaptor, regulate Git1 localization and this is dependent upon their interaction with the ubiquitin ligase Rsp5. Specifically, α-arrestin Aly2 stimulates Git1 trafficking to the vacuole under basal conditions, but in response to GPI-treatment, either Aly1 or Aly2 promote Git1 vacuole trafficking. Cell surface retention of Git1, as occurs in aly1∆ aly2∆ cells, is linked to impaired growth in the presence of exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of GPI somehow causes cellular toxicity. Regulation of αarrestin Aly1 by the protein phosphatase calcineurin improves steady-state and substrate-induced trafficking of Git1, however, calcineurin plays a larger role in Git1 trafficking beyond regulation of α-arrestins. Interestingly, loss of Aly1 and Aly2 increased phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole, and this was further exacerbated by GPI addition, suggesting that the effect is partially linked to Git1. Loss of Aly1 and Aly2 leads to increased incorporation of inositol label from [ 3 H]-inositol-labelled GPI into PI, confirming that internalized GPI influences PI balance and indicating a role for the a-arrestins in this regulation. Conclusions: The α-arrestins Aly1 and Aly2 are novel regulators of Git1 trafficking with previously unanticipated roles in controlling phospholipid distribution and balance.

Phosphatidylinositol (PI) is an essential phospholipid and critical component of membrane bilayers. The complete deacylation of PI by phospholipases of the B-type leads to the production of intracellular and extracellular glycerophosphoinositol (GPI), a water-soluble glycerophosphodiester. Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters that resides at the plasma membrane in yeast. Once internalized, GPI can be degraded to produce inositol, phosphate and glycerol, thereby contributing to reserves of these building blocks. Not surprisingly, GIT1 gene expression is controlled by nutrient balance, with limitation for phosphate or inositol each increasing GIT1 expression to facilitate GPI uptake. Less is known about how Git1 protein levels or localization are controlled. Here we show that the α-arrestins, an important class of protein trafficking adaptor, regulate the localization of Git1 in a manner dependent upon their association with the ubiquitin ligase Rsp5. Specifically, α-arrestin Aly2 is needed for effective Git1 internalization from the plasma membrane under basal conditions. However, in response to GPI-treatment of cells, either Aly1 or Aly2 can promote Git1 trafficking to the vacuole. Retention of Git1 at the cell surface, as occurs in aly1∆ aly2∆ cells, results in impaired growth in the presences of excess exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of this metabolite or its downstream products leads to cellular toxicity. We further show that regulation of α-arrestin Aly1 by the protein phosphatase calcineurin improves both steady-state and ligand-induced trafficking of Git1 when a mutant allele of Aly1 that mimics the dephosphorylated state at calcineurin-regulated residues is employed. Thus, calcineurin regulation of Aly1 is important for the GPI-ligand induced trafficking of Git1 by this α-arrestin, however, the role of calcineurin in regulating Git1 trafficking is much broader than can simply be explained by regulation of the α-arrestins. Finally, we find that loss of Aly1 and Aly2 leads to an increase in phosphatidylinositol-3-phosphate on the limiting membrane of the vacuole and this alteration is further exacerbated by addition of GPI, suggesting that the effect is at least partially linked to Git1 function. Indeed, loss of Aly1 and Aly2 leads to increased incorporation of inositol label from 3H-inositol-labelled GPI into PI, confirming that internalized GPI influences PI synthesis and indicating a role for the α-arrestins in regulating the process.

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.