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.     

Regulation of Fab1 phosphatidylinositol 3-phosphate 5-kinase pathway by Vac7 protein and Fig4, a polyphosphoinositide phosphatase family member

The Saccharomyces cerevisiae FAB1 gene encodes the sole phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinase responsible for synthesis of the polyphosphoinositide PtdIns(3,5)P(2). VAC7 encodes a 128-kDa transmembrane protein that localizes to vacuolar membranes. Both vac7 and fab1 null mutants have dramatically enlarged vacuoles and cannot grow at elevated temperatures. Additionally, vac7Delta mutants have nearly undetectable levels of PtdIns(3,5)P(2), suggesting that Vac7 functions to regulate Fab1 kinase activity. To test this hypothesis, we isolated a fab1 mutant allele that bypasses the requirement for Vac7 in PtdIns(3,5)P(2) production. Expression of this fab1 allele in vac7Delta mutant cells suppresses the temperature sensitivity, vacuolar morphology, and PtdIns(3,5)P(2) defects normally exhibited by vac7Delta mutants. We also identified a mutant allele of FIG4, whose gene product contains a Sac1 polyphosphoinositide phosphatase domain, which suppresses vac7Delta mutant phenotypes. Deletion of FIG4 in vac7Delta mutant cells suppresses the temperature sensitivity and vacuolar morphology defects, and dramatically restores PtdIns(3,5)P(2) levels. These results suggest that generation of PtdIns(3,5)P(2) by the Fab1 lipid kinase is regulated by Vac7, whereas turnover of PtdIns(3,5)P(2) is mediated in part by the Sac1 polyphosphoinositide phosphatase family member Fig4.     

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.     

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.     

Core protein machinery for mammalian phosphatidylinositol 3,5-bisphosphate synthesis and turnover that regulates the progression of endosomal transport. Novel Sac phosphatase joins the ArPIKfyve-PIKfyve complex

Perturbations in phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)-synthesizing enzymes result in enlarged endocytic organelles from yeast to humans, indicating evolutionarily conserved function of PtdIns(3,5)P2 in endosome-related events. This is reinforced by the structural and functional homology of yeast Vac14 and human Vac14 (ArPIKfyve), which activate yeast and mammalian PtdIns(3,5)P2-producing enzymes, Fab1 and PIKfyve, respectively. In yeast, PtdIns(3,5)P2-specific phosphatase, Fig4, in association with Vac14, turns over PtdIns(3,5)P2, but whether such a mechanism operates in mammalian cells and what the identity of mammalian Fig4 may be are unknown. Here we have identified and characterized Sac3, a Sac domain phosphatase, as the Fig4 mammalian counterpart. Endogenous Sac3, a widespread 97-kDa protein, formed a stable ternary complex with ArPIKfyve and PIKfyve. Concordantly, Sac3 cofractionated and colocalized with ArPIKfyve and PIKfyve. The intrinsic Sac3(WT) phosphatase activity preferably hydrolyzed PtdIns(3,5)P2 in vitro, although the other D5-phosphorylated polyphosphoinositides were also substrates. Ablation of endogenous Sac3 by short interfering RNAs elevated PtdIns(3,5)P2 in (32)P-labeled HEK293 cells. Ectopically expressed Sac3(WT) in COS cells colocalized with and dilated EEA1-positive endosomes, consistent with the PtdIns(3,5)P2 requirement in early endosome dynamics. In vitro reconstitution of carrier vesicle formation from donor early endosomes revealed a gain of function upon Sac3 loss, whereas PIKfyve or ArPIKfyve protein depletion produced a loss of function. These data demonstrate a coupling between the machinery for PtdIns(3,5)P2 synthesis and turnover achieved through a physical assembly of PIKfyve, ArPIKfyve, and Sac3. We suggest that the tight regulation in PtdIns(3,5)P2 homeostasis is mechanistically linked to early endosome dynamics in the course of cargo transport.     

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.     

Fab1p Is Essential for PtdIns(3)P 5-Kinase Activity and the Maintenance of Vacuolar Size and Membrane Homeostasis

The Saccharomyces cerevisiae FAB1 gene encodes a 257-kD protein that contains a cysteine-rich RING-FYVE domain at its NH₂-terminus and a kinase domain at its COOH terminus. Based on its sequence, Fab1p was initially proposed to function as a phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase (Yamamoto et al., 1995). Additional sequence analysis of the Fab1p kinase domain, reveals that Fab1p defines a subfamily of putative PtdInsP kinases that is distinct from the kinases that synthesize PtdIns(4,5)P₂. Consistent with this, we find that unlike wild-type cells, fab1Δ, fab1^tsf, and fab1 kinase domain point mutants lack detectable levels of PtdIns(3,5)P₂, a phosphoinositide recently identified both in yeast and mammalian cells. PtdIns(4,5)P₂ synthesis, on the other hand, is only moderately affected even in fab1Δ mutants. The presence of PtdIns(3)P in fab1 mutants, combined with previous data, indicate that PtdIns(3,5)P₂ synthesis is a two step process, requiring the production of PtdIns(3)P by the Vps34p PtdIns 3-kinase and the subsequent Fab1p- dependent phosphorylation of PtdIns(3)P yielding PtdIns(3,5)P₂. Although Vps34p-mediated synthesis of PtdIns(3)P is required for the proper sorting of hydrolases from the Golgi to the vacuole, the production of PtdIns(3,5)P₂ by Fab1p does not directly affect Golgi to vacuole trafficking, suggesting that PtdIns(3,5)P₂ has a distinct function. The major phenotypes resulting from Fab1p kinase inactivation include temperature-sensitive growth, vacuolar acidification defects, and dramatic increases in vacuolar size. Based on our studies, we hypothesize that whereas Vps34p is essential for anterograde trafficking of membrane and protein cargoes to the vacuole, Fab1p may play an important compensatory role in the recycling/turnover of membranes deposited at the vacuole. Interestingly, deletion of VAC7 also results in an enlarged vacuole morphology and has no detectable PtdIns(3,5)P₂, suggesting that Vac7p functions as an upstream regulator, perhaps in a complex with Fab1p. We propose that Fab1p and Vac7p are components of a signal transduction pathway which functions to regulate the efflux or turnover of vacuolar membranes through the regulated production of PtdIns(3,5)P₂.     

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.     

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.     

PtdIns(3,5)P2 finds a partner

Phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P(2)) is required for the sorting of a subset of membrane proteins at the late endosome. Unlike other phosphoinositides, binding partners for PtdIns(3,5)P(2) and its mechanism of action have not been characterized. New work by in this issue of Developmental Cell describes the identification of a yeast epsin-like protein that binds PtdIns(3,5)P(2) and functions in the transport of proteins through late endosomes to the lysosome-like vacuole.     

Phosphatidylinositol-3,5-bisphosphate: no longer the poor PIP2

Phosphoinositides play an important role in organelle identity by recruiting effector proteins to the host membrane organelle, thus decorating that organelle with molecular identity. Phosphatidylinositol-3,5-bisphos- phate [PtdIns(3,5)P(2) ] is a low-abundance phosphoinositide that predominates in endolysosomes in higher eukaryotes and in the yeast vacuole. Compared to other phosphoinositides such as PtdIns(4,5)P(2) , our understanding of the regulation and function of PtdIns(3,5)P(2) remained rudimentary until more recently. Here, we review many of the recent developments in PtdIns(3,5)P(2) function and regulation. PtdIns(3,5)P(2) is now known to espouse functions, not only in the regulation of endolysosome morphology, trafficking and acidification, but also in autophagy, signaling mediation in response to stresses and hormonal cues and control of membrane and ion transport. In fact, PtdIns(3,5)P(2) misregulation is now linked with several human neuropathologies including Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis. Given the functional versatility of PtdIns(3,5)P(2) , it is not surprising that regulation of PtdIns(3,5)P(2) metabolism is proving rather elaborate. PtdIns(3,5)P(2) synthesis and turnover are tightly coupled via a protein complex that includes the Fab1/PIKfyve lipid kinase and its antagonistic Fig4/Sac3 lipid phosphatase. Most interestingly, many PtdIns(3,5)P(2) regulators play simultaneous roles in its synthesis and turnover.     

PtdIns(3,5)P2 is required for delivery of endocytic cargo into the multivesicular body

The endocytic pathway transports cargo from the plasma membrane to early endosomes, where certain cargoes are sorted to the late endosome/multivesicular body. Biosynthetic cargo destined for the lysosome is also trafficked through the multivesicular body. Once delivered to the multivesicular body, cargo destined for the interior of the lysosome is selectively sorted into vesicles that bud into the lumen of the multivesicular body. These vesicles are released into the lumen of the lysosome upon the fusion of the multivesicular body and lysosomal limiting membranes. The yeast protein Fab1, which catalyzes the production of phosphatidylinositol (3,5) bisphosphate [PtdIns(3,5)P₂], is necessary for proper sorting of biosynthetic cargo in the multivesicular body. Utilizing an endocytosis screen, we isolated a novel allele of FAB1 that contains a point mutation in the lipid kinase domain. Characterization of this allele revealed reduced PtdIns(3,5)P₂ production, altered vacuole morphology, and biosynthetic protein sorting defects. We also found that endocytosis of the plasma membrane protein Ste3 is partially blocked downstream of the internalization step, and that delivery of the dye FM4-64 to the vacuole is delayed in fab1 mutants. Additionally, Ste3 is not efficiently sorted into multivesicular body vesicles in fab1 mutants and instead localizes to the vacuolar limiting membrane. These data show that PtdIns(3,5)P₂ is necessary for proper trafficking and sorting of endocytic cargo through the late endosome/multivesicular body.     

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.     

Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636

PIKfyve is an essential mammalian lipid kinase with pleiotropic cellular functions whose genetic knockout in mice leads to preimplantation lethality. Despite several reports for PIKfyve-catalyzed synthesis of phosphatidylinositol 5-phosphate (PtdIns5P) along with phosphatidylinositol-3,5-biphosphate [PtdIns(3,5)P(2)] in vitro and in vivo, the role of the PIKfyve pathway in intracellular PtdIns5P production remains underappreciated and the function of the PIKfyve-synthesized PtdIns5P pool poorly characterized. Hence, the recently discovered potent PIKfyve-selective inhibitor, the YM201636 compound, has been solely tested for inhibiting PtdIns(3,5)P(2) synthesis. Here, we have compared the in vitro and in vivo inhibitory potency of YM201636 toward PtdIns5P and PtdIns(3,5)P(2). Unexpectedly, we observed that at low doses (10-25 nM), YM201636 inhibited preferentially PtdIns5P rather than PtdIns(3,5)P(2) production in vitro, whereas at higher doses, the two products were similarly inhibited. In cellular contexts, YM201636 at 160 nM inhibited PtdIns5P synthesis twice more effectively compared with PtdIns(3,5)P(2) synthesis. In 3T3L1 adipocytes, human embryonic kidney 293 and Chinese hamster ovary (CHO-T) cells, levels of PtdIns5P dropped by 62-71% of the corresponding untreated controls, whereas those of PtdIns(3,5)P(2) fell by only 28-46%. The preferential inhibition of PtdIns5P versus PtdIns(3,5)P(2) at low doses of YM201636 was explored to probe contributions of the PIKfyve-catalyzed PtdIns5P pool to insulin-induced actin stress fiber disassembly in CHO-T cells, GLUT4 translocation in 3T3L1 adipocytes, and induction of aberrant cellular vacuolation in these or other cell types. The results provide the first experimental evidence that the principal pathway for PtdIns5P intracellular production is through PIKfyve and that insulin effect on actin stress fiber disassembly is mediated entirely by the PIKfyve-produced PtdIns5P pool.     

Expression of myotubularin by an adenoviral vector demonstrates its function as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase in muscle cell lines: involvement of PtdIns(3)P in insulin-stimulated glucose transport

X-linked myotubular myopathy is a muscle disorder caused by mutations on the myotubular myopathy-1 (MTM-1) gene, coding for myotubularin a 65-kDa polypeptide similar to protein phosphatases. Biochemical and in vivo studies define myotubularin as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase. To efficiently express myotubularin in muscle cell lines and adipocytes, we used an adenoviral genome recombinogenic to pcDNA3, and to other widely used expression vectors, to produce adenoviruses expressing wild-type (wt), catalytically inactive C375S, and substrate trap D278A myotubularin.[32P]Orthophosphate labeling followed by phosphoinositide analysis of differentiated L6 and C2C12 cells expressing myotubularin demonstrated increased PtdIns(3)P levels upon expression of the C375S and D278A mutants. In keeping with its biochemical function, overexpression of wt myotubularin as an enhanced green fluorescent protein fusion disrupted the endosomal punctuated staining of the FYVE (Fab1p/YOTB Vac1p/EEA1)-domain-containing PtdIns(3)P binding protein early endosomal antigen 1 as well as of a gluathione-S-transferase-FYVE probe directed to PtdIns(3)P. Expression of wt myotubularin, although not affecting activation of proximal insulin signal transduction targets such as protein kinase B and MAPK, induced a decrease in insulin-induced glucose uptake, whereas basal glucose uptake was augmented by expression of D278A (DA) and C375S (CS) mutants. Moreover, overexpression of myotubularin in 3T3-L1 adipocytes impaired insulin-induced translocation at the plasma membrane of green fluorescent protein-tagged glucose transporter 4. These data indicate that PtdIns(3)P is required to direct glucose transporter 4 to insulin-responsive compartments and/or to allow the translocation of the latter at the plasma membrane.We conclude that myotubularin, by modulating the intracellular levels of PtdIns(3)P, plays a role in the control of vesicular traffic related to glucose transport, by counteracting the activities of the PtdIns(3)P-producing phosphatidylinositol 3-kinases.     

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.     

Changing phosphoinositides “on the fly”: how trafficking vesicles avoid an identity crisis

Joining an antagonistic phosphoinositide (PtdInsP) kinase and phosphatase into a single protein complex may regulate rapid and local PtdInsP changes. This may be important for processes such as membrane fission that require a specific PtdInsP and that are innately local and rapid. Such a complex could couple vesicle formation, with erasing of the identity of the donor organelle from the vesicle prior to its fusion with target organelles, thus preventing organelle identity intermixing. Coordinating signals are postulated to switch the relative activities of the kinase and phosphatase in a spatio‐temporal manner that matches membrane fission events. The discovery of two such complexes supports this hypothesis. One regulates the interconversion of phosphatidylinositol and PtdIns(3)P by joining the Vps34 PtdIns 3‐kinase and the myotubularin 3‐phosphatases. The other regulates the interconversion between PtdIns(3)P and PtdIns(3,5)P₂ through the Fab1/PIKfyve kinase and the Fig4/mFig4 phosphatase. These lipids are essential components of the endosomal identity code.     

Myotubularin regulates the function of the late endosome through the gram domain-phosphatidylinositol 3,5-bisphosphate interaction

Myotubularin and related proteins constitute a large and highly conserved family possessing phosphoinositide 3-phosphatase activity, although not all members possess this activity. This family contains a conserved region called the GRAM domain that is found in a variety of proteins associated with membrane-coupled processes and signal transduction. Mutations of myotubularin are found in X-linked myotubular myopathy, a severe muscle disease. Mutations in the GRAM domain are responsible for this condition, suggesting crucial roles for this region. Here, we show that the GRAM domain of myotubularin binds to phosphoinositide with the highest affinity to phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)). In patients with myotubular myopathy, mutations in the myotubularin GRAM domain eliminate this binding, indicating that the PtdIns(3,5)P(2) binding ability of the GRAM (glucosyltransferases, Rablike GTPase activators and myotubularin) domain is crucial for the functions of myotubularin in vivo. Stimulation of epidermal growth factor recruits myotubularin to the late endosomal compartment in a manner dependent on the phosphoinositide binding. Overexpression of myotubularin inhibits epidermal growth factor receptor trafficking from late endosome to lysosome and induces the large endosomal vacuoles. Thus, our data suggest that myotubularin phosphatase physiologically functions in late endosomal trafficking and vacuolar morphology through interaction with PtdIns(3,5)P(2).     

The phosphoinositide kinase PIKfyve/Fab1p regulates terminal lysosome maturation in Caenorhabditis elegans

Membrane dynamics is necessary for cell homeostasis and signal transduction and is in part regulated by phosphoinositides. Pikfyve/Fab1p is a phosphoinositide kinase that phosphorylates phosphatidylinositol 3-monophosphate into phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and is implicated in membrane homeostasis in yeast and in mammalian cells. These two phosphoinositides are substrates of myotubularin phosphatases found mutated in neuromuscular diseases. We studied the roles of phosphatidylinositol phosphate kinase 3 (PPK-3), the orthologue of PIKfyve/Fab1p, in a multicellular organism, Caenorhabditis elegans. Complete loss of ppk-3 function induces developmental defects characterized by embryonic lethality, whereas partial loss of function leads to growth retardation. At the cellular level, ppk-3 mutants display a striking enlargement of vacuoles positive for lysosome-associated membrane protein 1 in different tissues. In the intestine, RAB-7-positive late endosomes are also enlarged. Membranes of the enlarged lysosomes originate at least in part from smaller lysosomes, and functional and genetic analyses show that the terminal maturation of lysosomes is defective. Protein degradation is not affected in the hypomorphic ppk-3 mutant and is thus uncoupled from membrane retrieval. We measured the level of PtdIns(3,5)P2 and showed that its production is impaired in this mutant. This work strongly suggests that the main function of PPK-3 is to mediate membrane retrieval from matured lysosomes through regulation of PtdIns(3,5)P2.     

Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires lumenal vacuolar hydrolase activities.

The Golgi/endosome-associated Vps34 phosphatidylinositol 3-kinase is essential for the sorting of hydrolases from the Golgi to the vacuole/lysosome. Upon inactivation of a temperature-conditional Vps34 kinase, cellular levels of PtdIns(3)P rapidly decrease and it has been proposed that this decrease is due to the continued turnover of PtdIns(3)P by cytoplasmic phosphatases. Here we show that mutations in VAM3 (vacuolar t-SNARE) and YPT7 (rab GTPase), which are required to direct protein and membrane delivery from prevacuolar endosomal compartments to the vacuole, dramatically increase/stabilize PtdIns(3)P levels in vivo by disrupting its turnover. We find that the majority of the total pool of PtdIns(3)P which has been synthesized, but not PtdIns(4)P, requires transport to the vacuole in order to be turned over. Unexpectedly, strains with impaired vacuolar hydrolase activity accumulate 4- to 5-fold higher PtdIns(3)P levels than wild-type cells, suggesting that lumenal vacuolar lipase and/or phosphatase activities degrade PtdIns(3)P. Because vacuolar hydrolases act in the lumen, PtdIns(3)P is likely to be transferred from the cytoplasmic membrane leaflet where it is synthesized, to the lumen of the vacuole. Interestingly, mutants that stabilize PtdIns(3)P accumulate small uniformly-sized vesicles (40-50 nm) within prevacuolar endosomes (multivesicular bodies) or the vacuole lumen. Based on these and other observations, we propose that PtdIns(3)P is degraded by an unexpected mechanism which involves the sorting of PtdIns(3)P into vesicles generated by invagination of the limiting membrane of the endosome or vacuole, ultimately delivering the phosphoinositide into the lumen of the compartment where it can be degraded by the resident hydrolases.