Atg18 Regulates Organelle Morphology and Fab1 Kinase Activity Independent of Its Membrane Recruitment by Phosphatidylinositol 3,5-Bisphosphate

Abstract

The lipid kinase Fab1 governs yeast vacuole homeostasis by generating PtdIns(3,5)P 2 on the vacuolar membrane. Recruitment of effector proteins by the phospholipid ensures precise regulation of vacuole morphology and function. Cells lacking the effector Atg18p have enlarged vacuoles and high PtdIns(3,5)P 2 levels. Although Atg18 colocalizes with Fab1p, it likely does not directly interact with Fab1p, as deletion of either kinase activator-VAC7 or VAC14-is epistatic to atg18⌬: atg18⌬vac7⌬ cells have no detectable PtdIns(3,5)P 2 . Moreover, a 2xAtg18 (tandem fusion) construct localizes to the vacuole membrane in the absence of PtdIns(3,5)P 2 , but requires Vac7p for recruitment. Like the endosomal PtdIns(3)P effector EEA1, Atg18 membrane binding may require a protein component. When the lipid requirement is bypassed by fusing Atg18 to ALP, a vacuolar transmembrane protein, vac14⌬ vacuoles regain normal morphology. Rescue is independent of PtdIns(3,5)P 2 , as mutation of the phospholipid-binding site in Atg18 does not prevent vacuole fission and properly regulates Fab1p activity. Finally, the vacuole-specific type-V myosin adapter Vac17p interacts with Atg18p, perhaps mediating cytoskeletal attachment during retrograde transport. Atg18p is likely a PtdIns(3,5)P 2 "sensor," acting as an effector to remodel membranes as well as regulating its synthesis via feedback that might involve Vac7p. INTRODUCTIONEukaryotic cells have evolved highly sophisticated macromolecular machinery to orchestrate the complex and dynamic process of membrane trafficking. These protein complexes vary widely in structure and function, but are all tightly coordinated in order to catalyze a sequence of specific events with the goal of transferring membrane cargo from one compartment to another. Despite the constant flux of membrane and proteins, organelles maintain distinct identities. To achieve the formation and maintenance of organelle size, shape, and function, trafficking complexes must be properly targeted and subsequently recycled (Munro, 2004;Behnia and Munro, 2005;Jordens et al., 2005). Lipids have been shown to play an increasingly important role in these processes, especially phosphoinositides (PtdInsPs).PtdInsPs are phosphorylated derivatives of phosphatidylinositol, of which there are seven species that can be rapidly interconverted by phosphorylation or dephosphorylation reactions. The occurrence of a specific PtdInsP transiently recruits cognate effector proteins that bind the PtdInsP via short modular motifs such as the FYVE and PH domains (Lemmon, 2003;Balla, 2005;Behnia and Munro, 2005;Di Paolo and De Camilli, 2006;Takenawa and Itoh, 2006). Hence, PtdInsPs are particularly suited to serve as membrane surface tags. Importantly, PtdInsP-binding modules differ greatly in their affinity and specificity for the various PtdInsPs, which when coupled with protein partners, generates a specificity code for each organelle (Lawe et al., 2000;Balla, 2005).PtdIns(3)P and PtdIns(3,5)P 2 , respectively, label early and late endosomal structu...

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

“…16 WIPI proteins (WIPI1 to WIPI4), mammalian homologs of yeast Atg18, may have the ability to sense and regulate PtdIns3P levels and participate in the initiation and maturation of the omegasome. 7,27,94,95 WDFY3 is implicated in cargo selection and delivery in selective autophagy. 17,27,96 It is further conjectured that the PtdIns3P synthesized in situ, together with its effector proteins, signals the de novo synthesis of other phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine), which constitute the autophagosomal membrane.…”

Assembly of a Fab1 Phosphoinositide Kinase Signaling Complex Requires the Fig4 Phosphoinositide Phosphatase

“…pJE181 (encoding Atg18), pBP74A, and pRB415A have been described previously (Efe et al, 2007). Constructs expressing FIG4 and fig4-1 were described in Gary et al (2002), and pRS416::FAB1 and pRS416::FAB1 D2134R were characterized in Gary et al (1998).…”

“…Vacuoles were labeled with the fluorescent probes N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide FM4-64 (Invitrogen, Carlsbad, CA) and/or 4-chloromethyl-7-aminocoumarin (Invitrogen) as described previously (Efe et al, 2007). Fluorescence and differential interference contrast (DIC) images of labeled cells and those expressing green fluorescent protein (GFP) fusion proteins were generated with a Delta Vision RT microscopy system (Applied Precision, Issaquah, WA).…”

“…Similar observations were obtained when these GFP-fusion proteins were expressed in a fab1⌬ strain (Supplemental Figure S1B). The vacuolar limiting membrane was labeled with the FM4-64 dye, and quantification of the fluorescence intensity was done as described previously (Figure 2, A and B; Efe et al, 2007). We used two methods to analyze the behavior of the GFP-fusion proteins: 1) fluorescence line plotting to determine fluorescence correlation between GFP and FM4-64 signal ( Figure 2A) and 2) quantification of the vacuole-to-cytosol (V/C) fluorescence ratio of the GFP signal ( Figure 2B).…”

“…Interestingly, recent work suggests that Fig4 is also necessary for the synthesis of PtdIns(3,5)P 2 ; unlike wild-type yeast cells, fig4⌬ cells do not exhibit a rapid and sharp increase in PtdIns(3,5)P 2 levels upon exposure to high salt (Dove et al, 1997;Duex et al, 2006a,b). Additionally, deletion of FIG4 prevents the steadystate increase in PtdIns(3,5)P 2 levels in atg18⌬ cells (Efe et al, 2007). Curiously, Vac14 is also implicated in turnover of PtdIns(3,5)P 2 (Duex et al, 2006b).…”