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

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.     

Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p

Phosphatidylinositol 3,5-bisphosphate (PtdIns[3,5]P(2)) was first identified as a non-abundant phospholipid whose levels increase in response to osmotic stress. In yeast, Fab1p catalyzes formation of PtdIns(3,5)P(2) via phosphorylation of PtdIns(3)P. We have identified Vac14p, a novel vacuolar protein that regulates PtdIns(3,5)P(2) 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)P(2) levels are regulated by Vac14p. Under basal conditions the levels of PtdIns(3,5)P(2) are 18-28-fold lower than the levels of PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P(2). After a 10 min exposure to hyperosmotic stress the levels of PtdIns(3,5)P(2) rise 20-fold, bringing it to a cellular concentration that is similar to the other phosphoinositides. This suggests that PtdIns(3,5)P(2) 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)P(2) continue to maintain a single large vacuole. These findings demonstrate that Vac14p regulates the levels of PtdIns(3,5)P(2) and provide insight into why PtdIns(3,5)P(2) levels rise in response to osmotic stress.     

Vacuole size control: regulation of PtdIns(3,5)P2 levels by the vacuole-associated Vac14-Fig4 complex, a PtdIns(3,5)P2-specific phosphatase

In the budding yeast Saccharomyces cerevisiae, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is synthesized by a single phosphatidylinositol 3-phosphate 5-kinase, Fab1. Cells deficient in PtdIns(3,5)P2 synthesis exhibit a grossly enlarged vacuole morphology, whereas increased levels of PtdIns(3,5)P2 provokes the formation of multiple small vacuoles, suggesting a specific role for PtdIns(3,5)P2 in vacuole size control. Genetic studies have indicated that Fab1 kinase is positively regulated by Vac7 and Vac14; deletion of either gene results in ablation of PtdIns(3,5)P2 synthesis and the formation of a grossly enlarged vacuole. More recently, a suppressor of vac7Delta mutants was identified and shown to encode a putative phosphoinositide phosphatase, Fig4. We demonstrate that Fig4 is a magnesium-activated PtdIns(3,5)P2-selective phosphoinositide phosphatase in vitro. Analysis of a Fig4-GFP fusion protein revealed that the Fig4 phosphatase is localized to the limiting membrane of the vacuole. Surprisingly, in the absence of Vac14, Fig4-GFP no longer localizes to the vacuole. However, Fig4-GFP remains localized to the grossly enlarged vacuoles of vac7 deletion mutants. Consistent with these observations, we found that Fig4 physically associates with Vac14 in a common membrane-associated complex. Our studies indicate that Vac14 both positively regulates Fab1 kinase activity and directs the localization/activation of the Fig4 PtdIns(3,5)P2 phosphatase.     

Hyperosmotic stress induces a rapid and transient increase in inositol 1,4,5-trisphosphate independent of abscisic acid in Arabidopsis cell culture

Phospholipid metabolism is involved in hyperosmotic-stress responses in plants. To investigate the role of phosphoinositide-specific phospholipase C (PI-PLC)-a key enzyme in phosphoinositide turnover-in hyperosmotic-stress signaling, we analyzed changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content in response to hyperosmotic shock or salinity in Arabidopsis thaliana T87 cultured cells. Within a few s, a hyperosmotic shock, caused by mannitol, NaCl, or dehydration, induced a rapid and transient increase in Ins(1,4,5)P3. However, no transient increase was detected in cells treated with ABA. Neomycin and U73122, inhibitors of PI-PLC, inhibited the increase in Ins(1,4,5)P3 caused by the hyperosmotic shock. A rapid increase in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in response to the hyperosmotic shock also occurred, but the rate of increase was much slower than that of Ins(1,4,5)P3. These findings indicate that the transient Ins(1,4,5)P3 production was due to the activation of PI-PLC in response to hyperosmotic stress. PI-PLC inhibitors also inhibited hyperosmotic stress-responsive expression of some dehydration-inducible genes, such as rd29A (lti78/cor78) and rd17 (cor47), that are controlled by the DRE/CRT cis-acting element but did not inhibit hyperosmotic stress-responsive expression of ABA-inducible genes, such as rd20. Taken together, these results suggest the involvement of PI-PLC and Ins(1,4,5)P3 in an ABA-independent hyperosmotic-stress signal transduction pathway in higher plants.     

Phosphatidylinositol 3,5‐bisphosphate regulates Ca2+ transport during yeast vacuolar fusion through the Ca2+ ATPase Pmc1

The transport of Ca²⁺ across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles under hyperosmotic stress become fragmented through fission events that requires the release of Ca²⁺ stores through the TRP channel Yvc1. This requires the phosphorylation of phosphatidylinositol‐3‐phosphate (PI3P) by the PI3P‐5‐kinase Fab1 to produce transient PI(3,5)P₂ pools. Ca²⁺ is also released during vacuole fusion upon trans‐SNARE complex assembly, however, its role remains unclear. The effect of PI(3,5)P₂ on Ca²⁺ flux during fusion was independent of Yvc1. Here, we show that while low levels of PI(3,5)P₂ were required for Ca²⁺ uptake into the vacuole, increased concentrations abolished Ca²⁺ efflux. This was as shown by the addition of exogenous dioctanoyl PI(3,5)P₂ or increased endogenous production of by the hyperactive fab1^T2250A mutant. In contrast, the lack of PI(3,5)P₂ on vacuoles from the kinase dead fab1^EEE mutant showed delayed and decreased Ca²⁺ uptake. The effects of PI(3,5)P₂ were linked to the Ca²⁺ pump Pmc1, as its deletion rendered vacuoles resistant to the effects of excess PI(3,5)P₂. Experiments with Verapamil inhibited Ca²⁺ uptake when added at the start of the assay, while adding it after Ca²⁺ had been taken up resulted in the rapid expulsion of Ca²⁺. Vacuoles lacking both Pmc1 and the H⁺/Ca²⁺ exchanger Vcx1 lacked the ability to take up Ca²⁺ and instead expelled it upon the addition of ATP. Together these data suggest that a balance of efflux and uptake compete during the fusion pathway and that the levels of PI(3,5)P₂ can modulate which path predominates.     

VAC14 nucleates a protein complex essential for the acute interconversion of PI3P and PI(3,5)P2 in yeast and mouse

The signalling lipid PI(3,5)P₂ is generated on endosomes and regulates retrograde traffic to the trans‐Golgi network. Physiological signals regulate rapid, transient changes in PI(3,5)P₂ levels. Mutations that lower PI(3,5)P₂ cause neurodegeneration in human patients and mice. The function of Vac14 in the regulation of PI(3,5)P₂ was uncharacterized previously. Here, we predict that yeast and mammalian Vac14 are composed entirely of HEAT repeats and demonstrate that Vac14 exerts an effect as a scaffold for the PI(3,5)P₂ regulatory complex by direct contact with the known regulators of PI(3,5)P₂: Fig4, Fab1, Vac7 and Atg18. We also report that the mouse mutant ingls (in fantile gl ios is) results from a missense mutation in Vac14 that prevents the association of Vac14 with Fab1, generating a partial complex. Analysis of ingls and two additional mutants provides insight into the organization of the PI(3,5)P₂ regulatory complex and indicates that Vac14 mediates three distinct mechanisms for the rapid interconversion of PI3P and PI(3,5)P₂. Moreover, these studies show that the association of Fab1 with the complex is essential for viability in the mouse.     

Elevating PI3P drives select downstream membrane trafficking pathways

Phosphoinositide signaling lipids are essential for several cellular processes. The requirement for a phosphoinositide is conventionally studied by depleting the corresponding lipid kinase. However, there are very few reports on the impact of elevating phosphoinositides. That phosphoinositides are dynamically elevated in response to stimuli suggests that, in addition to being required, phosphoinositides drive downstream pathways. To test this hypothesis, we elevated the levels of phosphatidylinositol-3-phosphate (PI3P) by generating hyperactive alleles of the yeast phosphatidylinositol 3-kinase, Vps34. We find that hyperactive Vps34 drives certain pathways, including phosphatidylinositol-3,5-bisphosphate synthesis and retrograde transport from the vacuole. This demonstrates that PI3P is rate limiting in some pathways. Interestingly, hyperactive Vps34 does not affect endosomal sorting complexes required for transport (ESCRT) function. Thus, elevating PI3P does not always increase the rate of PI3P-dependent pathways. Elevating PI3P can also delay a pathway. Elevating PI3P slowed late steps in autophagy, in part by delaying the disassembly of autophagy proteins from mature autophagosomes as well as delaying fusion of autophagosomes with the vacuole. This latter defect is likely due to a more general defect in vacuole fusion, as assessed by changes in vacuole morphology. These studies suggest that stimulus-induced elevation of phosphoinositides provides a way for these stimuli to selectively regulate downstream processes.     

Inactivation and dephosphorylation of protein kinase Balpha (PKBalpha) promoted by hyperosmotic stress.

To study the role of protein kinase B (PKB) in response to cellular stress, we examined PKBalpha activity following different stress treatments. Hyperosmotic but not chemical stress resulted in inactivation of PKBalpha and prevented activation by pervanadate and mitogens. Hyperosmotic shock did not affect the MAP kinase pathway, suggesting that this inhibitory effect was specific for PKB. Our data further indicate that downregulation occurs via dephosphorylation of Thr308 and Ser473, the major regulatory phosphorylation sites of PKBalpha. Indeed, calyculin A, which inhibits protein phosphatases 1 and 2A, effectively blocked hyperosmotic stress-mediated inactivation (dephosphorylation) of PKBalpha. High osmolarity did not affect phosphatidylinositol 3-kinase activity but led to a marked increase in PI(3,4,5)P3 and a decrease in PI(3,4)P2 formation after pervanadate stimulation, suggesting that hyperosmotic stress has an inhibitory effect on a phosphatidylinositol 5-phosphatase which converts PI(3,4,5)P3 into PI(3,4)P2. Immunofluorescence studies revealed that membrane translocation, a prerequisite for PKB activation, was not affected by hyperosmotic stress. Our results indicate that hyperosmotic stress can act at two levels: (i) inhibition of phosphorylation of Thr308 and Ser473 by upstream kinases and (ii) by promoting rapid dephosphorylation of these regulatory sites.     

Assembly of a Fab1 phosphoinositide kinase signaling complex requires the Fig4 phosphoinositide phosphatase

Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P₂] regulates several vacuolar functions, including acidification, morphology, and membrane traffic. The lipid kinase Fab1 converts phosphatidylinositol-3-phosphate [PtdIns(3)P] to PtdIns(3,5)P₂. PtdIns(3,5)P₂ levels are controlled by the adaptor-like protein Vac14 and the Fig4 PtdIns(3,5)P₂-specific 5-phosphatase. Interestingly, Vac14 and Fig4 serve a dual function: they are both implicated in the synthesis and turnover of PtdIns(3,5)P₂ by an unknown mechanism. We now show that Fab1, through its chaperonin-like domain, binds to Vac14 and Fig4 and forms a vacuole-associated signaling complex. The Fab1 complex is tethered to the vacuole via an interaction between the FYVE domain in Fab1 and PtdIns(3)P on the vacuole. Moreover, Vac14 and Fig4 bind to each other directly and are mutually dependent for interaction with the Fab1 kinase. Our observations identify a protein complex that incorporates the antagonizing Fab1 lipid kinase and Fig4 lipid phosphatase into a common functional unit. We propose a model explaining the dual roles of Vac14 and Fig4 in the synthesis and turnover of PtdIns(3,5)P₂.     

Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), made by Fab1p, is essential for vesicle recycling from vacuole/lysosomal compartments and for protein sorting into multivesicular bodies. To isolate PtdIns(3,5)P2 effectors, we identified Saccharomyces cerevisiae mutants that display fab1delta-like vacuole enlargement, one of which lacked the SVP1/YFR021w/ATG18 gene. Expressed Svp1p displays PtdIns(3,5)P2 binding of exquisite specificity, GFP-Svp1p localises to the vacuole membrane in a Fab1p-dependent manner, and svp1delta cells fail to recycle a marker protein from the vacuole to the Golgi. Cells lacking Svp1p accumulate abnormally large amounts of PtdIns(3,5)P2. These observations identify Svp1p as a PtdIns(3,5)P2 effector required for PtdIns(3,5)P2-dependent membrane recycling from the vacuole. Other Svp1p-related proteins, including human and Drosophila homologues, bind PtdIns(3,5)P2 similarly. Svp1p and related proteins almost certainly fold as beta-propellers, and the PtdIns(3,5)P2-binding site is on the beta-propeller. It is likely that many of the Svp1p-related proteins that are ubiquitous throughout the eukaryotes are PtdIns(3,5)P2 effectors. Svp1p is not involved in the contributions of FAB1/PtdIns(3,5)P2 to MVB sorting or to vacuole acidification and so additional PtdIns(3,5)P2 effectors must exist.     

Vac14 controls PtdIns(3,5)P(2) synthesis and Fab1-dependent protein trafficking to the multivesicular body

BACKGROUND: The PtdIns3P 5-kinase Fab1 makes PtdIns(3,5)P(2), a phosphoinositide essential for retrograde trafficking between the vacuole/lysosome and the late endosome and also for trafficking of some proteins into the vacuole via multivesicular bodies (MVB). No regulators of Fab1 were identified until recently. RESULTS: Visual screening of the Eurofan II panel of S. cerevisiae deletion mutants identified YLR386w as a novel regulator of vacuolar function. Others recently identified this ORF as encoding the vacuolar inheritance gene VAC14. Like fab1 mutants, yeast lacking Vac14 have enlarged vacuoles that do not acidify correctly. FAB1 overexpression corrects these defects. vac14Delta cells make very little PtdIns(3,5)P(2), and hyperosmotic shock does not stimulate PtdIns(3,5)P(2) synthesis in the normal manner, implicating Vac14 in Fab1 regulation. We also show that, like fab1Delta mutants, vac14Delta cells fail to sort GFP-Phm5 to the MVB and thence to the vacuole: irreversible ubiquitination of GFP-Phm5 overcomes this defect. In the BY4742 genetic background, loss of Vac14 causes much more penetrant effects on phosphoinositide metabolism and vacuolar trafficking than does loss of Vac7, another regulator of Fab1. Vac14 contains motifs suggestive of a role in protein trafficking and interacts with several proteins involved in clathrin-mediated membrane sorting and phosphoinositide metabolism. CONCLUSIONS: Vac14 and Vac7 are both upstream activators of Fab1-catalysed PtdIns(3,5)P(2) synthesis, with Vac14 the dominant contributor to the hierarchy of control. Vac14 is essential for the regulated synthesis of PtdIns(3,5)P(2), for control of trafficking of some proteins to the vacuole lumen via the MVB, and for maintenance of vacuole size and acidity.     

Atg18 regulates organelle morphology and Fab1 kinase activity independent of its membrane recruitment by phosphatidylinositol 3,5-bisphosphate

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 atg18Delta: atg18Deltavac7Delta 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, vac14Delta 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.     

Weak acid and alkali stress regulate phosphatidylinositol bisphosphate synthesis in Saccharomyces cerevisiae

Weak organic acids are used as food preservatives to inhibit the growth of spoilage yeasts, including Saccharomyces cerevisiae. Long-term adaptation to weak acids requires the increased expression of the ATP-binding cassette transporter Pdr12p, which catalyses the active efflux of the weak acids from the cytosol; however, very little is known about the signalling events immediately following application of weak acid stress. We have investigated the effects of weak acids on two stress-responsive signalling molecules, PtdIns(3,5)P2 and PtdIns(4,5)P2, which in S. cerevisiae are synthesized by Fab1p and Mss4p respectively. At low extracellular pH, benzoic acid, sorbic acid and acetic acid all cause a transient reduction in PtdIns(3,5)P2 accumulation and a more persistent rise in PtdIns(4,5)P2 levels. The increase in PtdIns(4,5)P2 levels is accompanied by a reorganization of the actin cytoskeleton. However, changes in PtdInsP2 levels are independent of weak acid-induced Pdr12p expression. In contrast, changing the extracellular medium to alkaline pH provokes a prolonged and substantial rise in PtdIns(3,5)P2 levels. As PtdIns(3,5)P2 synthesis is required for correct vacuole acidification, it is possible that levels of this molecule are modulated to maintain intracellular pH homoeostasis in response to weak acid and alkali stresses. In conclusion, we have expanded the repertoire of stress responses that affect PtdInsP2 levels to include weak acid and alkali stresses.     

Salt-stress-induced association of phosphatidylinositol 4,5-bisphosphate with clathrin-coated vesicles in plants

Plants exposed to hyperosmotic stress undergo changes in membrane dynamics and lipid composition to maintain cellular integrity and avoid membrane leakage. Various plant species respond to hyperosmotic stress with transient increases in PtdIns(4,5)P(2); however, the physiological role of such increases is unresolved. The plasma membrane represents the outermost barrier between the symplast of plant cells and its apoplastic surroundings. In the present study, the spatio-temporal dynamics of stress-induced changes in phosphoinositides were analysed in subcellular fractions of Arabidopsis leaves to delineate possible physiological roles. Unlabelled lipids were separated by TLC and quantified by gas-chromatographic detection of associated fatty acids. Transient PtdIns(4,5)P(2) increases upon exposure to hyperosmotic stress were detected first in enriched plasmamembrane fractions, however, at later time points, PtdIns(4,5)P(2) was increased in the endomembrane fractions of the corresponding two-phase systems. When major endomembranes were enriched from rosette leaves prior to hyperosmotic stress and during stimulation for 60 min, no stress-induced increases in the levels of PtdIns(4,5)P(2) were found in fractions enriched for endoplasmic reticulum, nuclei or plastidial membranes. Instead, increased PtdIns(4,5)P(2) was found in CCVs (clathrin-coated vesicles), which proliferated several-fold in mass within 60 min of hyperosmotic stress, according to the abundance of CCV-associated proteins and lipids. Monitoring the subcellular distribution of fluorescence-tagged reporters for clathrin and PtdIns(4,5)P(2) during transient co-expression in onion epidermal cells indicates rapid stress-induced co-localization of clathrin with PtdIns(4,5)P(2) at the plasma membrane. The results indicate that PtdIns(4,5)P(2) may act in stress-induced formation of CCVs in plant cells, highlighting the evolutionary conservation of the phosphoinositide system between organismic kingdoms.     

Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P(2) and PI(5)P

Normal steady-state levels of the signalling lipids PI(3,5)P(2) and PI(5)P require the lipid kinase FAB1/PIKfyve and its regulators, VAC14 and FIG4. Mutations in the PIKfyve/VAC14/FIG4 pathway are associated with Charcot-Marie-Tooth syndrome and amyotrophic lateral sclerosis in humans, and profound neurodegeneration in mice. Hence, tight regulation of this pathway is critical for neural function. Here, we examine the localization and physiological role of VAC14 in neurons. We report that endogenous VAC14 localizes to endocytic organelles in fibroblasts and neurons. Unexpectedly, VAC14 exhibits a pronounced synaptic localization in hippocampal neurons, suggesting a role in regulating synaptic function. Indeed, the amplitude of miniature excitatory postsynaptic currents is enhanced in both Vac14(-/-) and Fig4(-/-) neurons. Re-introduction of VAC14 in postsynaptic Vac14(-/-) cells reverses this effect. These changes in synaptic strength in Vac14(-/-) neurons are associated with enhanced surface levels of the AMPA-type glutamate receptor subunit GluA2, an effect that is due to diminished regulated endocytosis of AMPA receptors. Thus, VAC14, PI(3,5)P(2) and/or PI(5)P play a role in controlling postsynaptic function via regulation of endocytic cycling of AMPA receptors.     

Vac14 Protein Multimerization Is a Prerequisite Step for Fab1 Protein Complex Assembly and Function

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) helps control various endolysosome functions including organelle morphology, membrane recycling, and ion transport. Further highlighting its importance, PtdIns(3,5)P₂ misregulation leads to the development of neurodegenerative diseases like Charcot-Marie-Tooth disease. The Fab1/PIKfyve lipid kinase phosphorylates PtdIns(3)P into PtdIns(3,5)P₂ whereas the Fig4/Sac3 lipid phosphatase antagonizes this reaction. Interestingly, Fab1 and Fig4 form a common protein complex that coordinates synthesis and degradation of PtdIns(3,5)P₂ by a poorly understood process. Assembly of the Fab1 complex requires Vac14/ArPIKfyve, a multimeric scaffolding adaptor protein that coordinates synthesis and turnover of PtdIns(3,5)P₂. However, the properties and function of Vac14 multimerization remain mostly uncharacterized. Here we identify several conserved C-terminal motifs on Vac14 required for self-interaction and provide evidence that Vac14 likely forms a dimer. We also show that monomeric Vac14 mutants do not support interaction with Fab1 or Fig4, suggesting that Vac14 multimerization is likely the first molecular event in the assembly of the Fab1 complex. Finally, we show that cells expressing monomeric Vac14 mutants have enlarged vacuoles that do not fragment after hyperosmotic shock, which indicates that PtdIns(3,5)P₂ levels are greatly abated. Therefore, our observations support an essential role for the Vac14 homocomplex in controlling PtdIns(3,5)P₂ levels.     

Phosphatidylinositol 4,5-bisphosphate regulates two steps of homotypic vacuole fusion

Yeast vacuoles undergo cycles of fragmentation and fusion as part of their transmission to the daughter cell and in response to changes of nutrients and the environment. Vacuole fusion can be reconstituted in a cell free system. We now show that the vacuoles synthesize phosphoinositides during in vitro fusion. Of these phosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are important for fusion. Monoclonal antibodies to PI(4,5)P(2), neomycin (a phosphoinositide ligand), and phosphatidylinositol-specific phospholipase C interfere with the reaction. Readdition of PI(4, 5)P(2) restores fusion in each case. Phosphatidylinositol 3-phosphate and PI(3,5)P(2) synthesis are not required. PI(4,5)P(2) is necessary for priming, i.e., for the Sec18p (NSF)-driven release of Sec17p (alpha-SNAP), which activates the vacuoles for subsequent tethering and docking. Therefore, it represents the kinetically earliest requirement identified for vacuole fusion so far. Furthermore, PI(4,5)P(2) is required at a step that can only occur after docking but before the BAPTA sensitive step in the latest stage of the reaction. We hence propose that PI(4,5)P(2) controls two steps of vacuole fusion.     

Complementation analysis in PtdInsP kinase-deficient yeast mutants demonstrates that Schizosaccharomyces pombe and murine Fab1p homologues are phosphatidylinositol 3-phosphate 5-kinases

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) is widespread in eukaryotic cells. In Saccharomyces cerevisiae, PtdIns(3,5)P(2) synthesis is catalyzed by the PtdIns3P 5-kinase Fab1p, and loss of this activity results in vacuolar morphological defects, indicating that PtdIns(3,5)P(2) is essential for vacuole homeostasis. We have therefore suggested that all Fab1p homologues may be PtdIns3P 5-kinases involved in membrane trafficking. It is unclear which phosphatidylinositol phosphate kinases (PIPkins) are responsible for PtdIns(3,5)P(2) synthesis in higher eukaryotes. To clarify how PtdIns(3,5)P(2) is synthesized in mammalian and other cells, we determined whether yeast and mammalian Fab1p homologues or mammalian Type I PIPkins (PtdIns4P 5-kinases) make PtdIns(3,5)P(2) in vivo. The recently cloned murine (p235) and Schizosaccharomyces pombe FAB1 homologues both restored basal PtdIns(3,5)P(2) synthesis in Deltafab1 cells and made PtdIns(3,5)P(2) in vitro. Only p235 corrected the growth and vacuolar defects of fab1 S. cerevisiae. A mammalian Type I PIPkin supported no PtdIns(3,5)P(2) synthesis. Thus, FAB1 and its homologues constitute a distinct class of Type III PIPkins dedicated to PtdIns(3,5)P(2) synthesis. The differential abilities of p235 and of SpFab1p to complement the phenotypic defects of Deltafab1 cells suggests that interaction(s) with other protein factors may be important for spatial and/or temporal regulation of PtdIns(3,5)P(2) synthesis. These results also suggest that p235 may regulate a step in membrane trafficking in mammalian cells that is analogous to its function in yeast.