Alpha-arrestins Aly1/Art6 and Aly2/Art3 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis.

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 α-arrestin 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. SIGNIFICANCE: To our knowledge, this is the first example of α-arrestin regulation of phosphatidyliniositol-3-phosphate levels. In future studies it will be exciting to determine if other α-arrestins similarly alter PI and PIPs to change the cellular landscape.

Septin-coated microtubules promote maturation of multivesicular bodies by inhibiting their motility.

The microtubule cytoskeleton consists of microtubule subsets with distinct compositions of microtubule-associated proteins, which instruct the position and traffic of subcellular organelles. In the endocytic pathway, these microtubule-associated cues are poorly understood. Here, we report that in MDCK cells, endosomes with multivesicular body (MVB) and late endosome (LE) markers localize preferentially to microtubules coated with septin GTPases. Compared with early endosomes, CD63-containing MVBs/LEs are largely immotile on septin-coated microtubules. In vitro reconstitution assays revealed that the motility of isolated GFP-CD63 endosomes is directly inhibited by microtubule-associated septins. Quantification of CD63-positive endosomes containing the early endosome antigen (EEA1), the Rab7 effector and dynein adaptor RILP or Rab27a, showed that intermediary EEA1- and RILP-positive GFP-CD63 preferentially associate with septin-coated microtubules. Septin knockdown enhanced GFP-CD63 motility and decreased the percentage of CD63-positive MVBs/LEs with lysobiphosphatidic acid without impacting the fraction of EEA1-positive CD63. These results suggest that MVB maturation involves immobilization on septin-coated microtubules, which may facilitate multivesiculation and/or organelle-organelle contacts.

A septin GTPase scaffold of dynein-dynactin motors triggers retrograde lysosome transport.

The metabolic and signaling functions of lysosomes depend on their intracellular positioning and trafficking, but the underlying mechanisms are little understood. Here, we have discovered a novel septin GTPase-based mechanism for retrograde lysosome transport. We found that septin 9 (SEPT9) associates with lysosomes, promoting the perinuclear localization of lysosomes in a Rab7-independent manner. SEPT9 targeting to mitochondria and peroxisomes is sufficient to recruit dynein and cause perinuclear clustering. We show that SEPT9 interacts with both dynein and dynactin through its GTPase domain and N-terminal extension, respectively. Strikingly, SEPT9 associates preferentially with the dynein intermediate chain (DIC) in its GDP-bound state, which favors dimerization and assembly into septin multimers. In response to oxidative cell stress induced by arsenite, SEPT9 localization to lysosomes is enhanced, promoting the perinuclear clustering of lysosomes. We posit that septins function as GDP-activated scaffolds for the cooperative assembly of dynein-dynactin, providing an alternative mechanism of retrograde lysosome transport at steady state and during cellular adaptation to stress.