The Vac14p–Fig4p complex acts independently of Vac7p and couples PI3,5P2 synthesis and turnover

Abstract

Phosphoinositide-signaling lipids function in diverse cellular pathways. Dynamic changes in the levels of these signaling lipids regulate multiple processes. In particular, when Saccharomyces cerevisiae cells are exposed to hyperosmotic shock, PI3,5P2 (phosphatidylinositol [PI] 3,5-bisphosphate) levels transiently increase 20-fold. This causes the vacuole to undergo multiple acute changes. Control of PI3,5P2 levels occurs through regulation of both its synthesis and turnover. Synthesis is catalyzed by the PI3P 5-kinase Fab1p, and turnover is catalyzed by the PI3,5P2 5-phosphatase Fig4p. In this study, we show that two putative Fab1p activators, Vac7p and Vac14p, independently regulate Fab1p activity. Although Vac7p only regulates Fab1p, surprisingly, we find that Vac14 regulates both Fab1p and Fig4p. Moreover, Fig4p itself functions in both PI3,5P2 synthesis and turnover. In both the absence and presence of Vac7p, the Vac14p–Fig4p complex controls the hyperosmotic shock–induced increase in PI3,5P2 levels. These findings suggest that the dynamic changes in PI3,5P2 are controlled through a tight coupling of synthesis and turnover.

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

“…Together, these data suggest that Fab1 has a direct role in targeting Vac14 and Fig4 to the vacuole, perhaps via interactions with the CCT and CCR domains. (Rudge et al, 2004;Duex et al, 2006b). Indeed, we observed coIP of Vac14-HA and Fig4 ( Figure 5A).…”

“…Conceivably, Fig4 may stimulate PtdIns(3,5)P 2 synthesis by mediating direct protein-protein interactions or through its phosphatase activity, possibly by acting as a protein phosphatase (Duex et al, 2006b). To differentiate between these possibilities, we used a catalytic-inactive FIG4 allele, fig4-1, which was initially isolated as a suppressor of the vac7⌬ phenotype Rudge et al, 2004).…”

“…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). A key question is how Vac14 and Fig4 carry out their dual roles in PtdIns(3,5)P 2 synthesis and turnover?…”

“…Conceivably, these proteins form an intricate network of interactions that regulate PtdIns(3,5)P 2 . In yeast, the only established interaction as of yet is between Fig4 and Vac14 (Rudge et al, 2004;Duex et al, 2006b), whereas assays testing for Fab1 interaction with its regulators have thus far been unsuccessful Rudge et al, 2004). In mammals, it is proposed that PIKfyve forms a ternary complex with ArPIKfyve and hSac3, the Vac14 and Fig4 orthologues, respectively (Sbrissa et al, 2007).…”

rSac3, a novel Sac domain phosphoinositide phosphatase, promotes neurite outgrowth in PC12 cells

“…It interacts with Vac14p and forms the Vac14p-Fig4p complex. This complex controls the hyperosmotic shock-induced increase in PI(3,5)P 2 levels to maintain the vacuole size [29,30]. hSac1 localizes to the ER and Golgi apparatus, and could interact with members of the COP I complex through its Cterminal KXKXXX motif [23].…”