Phosphatidylinositol 3,5-bisphosphate (PtdIns[3,5]P2) was first identified as a nonabundant phospholipid whose levels increase in response to osmotic stress. In yeast, Fab1p catalyzes formation of PtdIns(3,5)P2 via phosphorylation of PtdIns(3)P. We have identified Vac14p, a novel vacuolar protein that regulates PtdIns(3,5)P2 synthesis by modulating Fab1p activity in both the absence and presence of osmotic stress. We find that PtdIns(3)P levels are also elevated in response to osmotic stress, yet, only the elevation of PtdIns(3,5)P2 levels are regulated by Vac14p. Under basal conditions the levels of PtdIns(3,5)P2 are 18–28-fold lower than the levels of PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P2. After a 10 min exposure to hyperosmotic stress the levels of PtdIns(3,5)P2 rise 20-fold, bringing it to a cellular concentration that is similar to the other phosphoinositides. This suggests that PtdIns(3,5)P2 plays a major role in osmotic stress, perhaps via regulation of vacuolar volume. In fact, during hyperosmotic stress the vacuole morphology of wild-type cells changes dramatically, to smaller, more highly fragmented vacuoles, whereas mutants unable to synthesize PtdIns(3,5)P2 continue to maintain a single large vacuole. These findings demonstrate that Vac14p regulates the levels of PtdIns(3,5)P2 and provide insight into why PtdIns(3,5)P2 levels rise in response to osmotic stress.

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The signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), likely functions in multiple signaling pathways. Here, we report the characterization of a mouse mutant lacking Vac14, a regulator of PI(3,5)P 2 synthesis. The mutant mice exhibit massive neurodegeneration, particularly in the midbrain and in peripheral sensory neurons. Cell bodies of affected neurons are vacuolated, and apparently empty spaces are present in areas where neurons should be present. Similar vacuoles are found in cultured neurons and fibroblasts. Selective membrane trafficking pathways, especially endosome-to-TGN retrograde trafficking, are defective. This report, along with a recent report on a mouse with a null mutation in Fig4, presents the unexpected finding that the housekeeping lipid, PI(3,5)P 2, is critical for the survival of neural cells.T he low-abundance signaling lipids, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P 2 ) and phosphatidylinositol 5-phosphate (PI(5)P), were discovered relatively recently (1-3). Because of their low abundance and the limited number of tools available for their study, relatively little is known about these lipids.An interesting property of PI(3,5)P 2 occurs in yeast, where a stimulus of hyperosmotic shock induces dramatic and transient changes in the levels of PI(3,5)P 2 . The levels of PI(3,5)P 2 transiently rise Ͼ20-fold (4). Within 1 minute, the levels rise 5-fold; by 5 minutes, they increase Ͼ20-fold; there is a short plateau of 10 min, and then PI(3,5)P 2 levels decrease at a rate similar to their increase. The rapid decrease in PI(3,5)P 2 levels occurs even though the cells remain in hyperosmotic media. Vacuole volume undergoes transient changes that parallel PI(3,5)P 2 levels. That these rapid and transient changes occur even in the presence of a sustained stimulus strongly suggests that PI(3,5)P 2 plays a major role in signaling pathways related to adaptation.Several proteins are required for the synthesis and turnover of PI(3,5)P 2 . PI(3,5)P 2 is synthesized from PI(3)P by the PI(3)P 5-kinase Fab1/PIKfyve/PIP5K3 (5, 6). Fab1 is stimulated by a regulatory complex that contains Vac14 (7, 8) and Fig4 (4, 9). Surprisingly, the Vac14/Fig4 complex plays two opposing roles in the regulation of steady-state levels of PI(3,5)P 2 . Vac14/Fig4 both activate Fab1 and also function in the breakdown of PI(3,5)P 2 through the lipid phosphatase activity of Fig4 (4, 9-11).In mammals, generation of PI(3,5)P 2 is predicted to impact PI(5)P production. In vitro studies have shown that PI(5)P can be generated from PI(3,5)P 2 through the PI(3,5)P 2 3-phosphatase activity of members of the myotubularin family and related proteins including MTM1, MTMR1, MTMR2, MTMR3, MTMR6, and hJUMPY/MTMR14 (12-15). In addition, PIKfyve/Fab1 can generate both PI(3,5)P 2 and PI(5)P in vitro (16). The source of PI(5)P in vivo has not been established. However, the generation of PI(5)P from either pathway requires PIKfyve/ Fab1 activity, either to produce the substrate for myotubularin [PI(3,5)P 2 ] or to produce PI(5...

Phosphoinositide lipids regulate complex events via the recruitment of proteins to a specialized region of the membrane at a specific time. Precise control of both the synthesis and turnover of phosphoinositide lipids is integral to membrane trafficking, signal transduction, and cytoskeletal rearrangements. Little is known about the acute regulation of the levels of these signaling lipids. When Saccharomyces cerevisiae cells are treated with hyperosmotic medium the levels of phosphatidylinositol 3,5-bisphosphate (PI3,5P 2 ) increase 20-fold. Here we show that this 20-fold increase is rapid and occurs within 5 min. Surprisingly, these elevated levels are transient. Fifteen minutes following hyperosmotic shock they decrease at a rapid rate, even though the cells remain in hyperosmotic medium. In parallel with the rapid increase in the levels of PI3,5P 2 , vacuole volume decreases rapidly. Furthermore, concomitant with a return to basal levels of PI3,5P 2 vacuole volume is restored. We show that Fig4p, consistent with its proposed role as a PI3,5P 2 5-phosphatase, is required in vivo for this rapid return to basal levels of PI3,5P 2 . Surprisingly, we find that Fig4p is also required for the hyperosmotic shock-induced increase in PI3,5P 2 levels. These findings demonstrate that following hyperosmotic shock, large, transient changes occur in the levels of PI3,5P 2 and further suggest that Fig4p is important in regulating both the acute rise and subsequent fall in PI3,5P 2 levels.

Normal cellular function requires that organelles be positioned in specific locations. The direction in which molecular motors move organelles is based in part on the polarity of microtubules and actin filaments. However, this alone does not determine the intracellular destination of organelles. For example, the yeast class V myosin, Myo2p, moves several organelles to distinct locations during the cell cycle. Thus the movement of each type of Myo2p cargo must be regulated uniquely. Here we report a regulatory mechanism that specifically provides directionality to vacuole movement. The vacuole-specific Myo2p receptor, Vac17p, has a key function in this process. Vac17p binds simultaneously to Myo2p and to Vac8p, a vacuolar membrane protein. The transport complex, Myo2p-Vac17p-Vac8p, moves the vacuole to the bud, and is then disrupted through the degradation of Vac17p. The vacuole is ultimately deposited near the centre of the bud. Removal of a PEST sequence (a potential signal for rapid protein degradation) within Vac17p causes its stabilization and the subsequent 'backward' movement of vacuoles, which mis-targets them to the neck between the mother cell and the bud. Thus the regulated disruption of this transport complex places the vacuole in its proper location. This may be a general mechanism whereby organelles are deposited at their terminal destination.