An intramolecular interaction within the lipid kinase Fab1 regulates cellular phosphatidylinositol 3,5-bisphosphate lipid levels

Lang, Michael J.; Strunk, Bethany S.; Azad, Nadia; Petersen, Jason L.; Weisman, Lois S.

doi:10.1091/mbc.e16-06-0390

Cited by 16 publications

(19 citation statements)

References 34 publications

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“…The levels of PI(3,5)P 2 , as well as those of its precursor PI3P and product PI5P, dynamically change upon specific stresses. Moreover dominant active mutations in Fab1 and PIKfyve have been isolated and characterized (Duex et al , 2006b; Lang et al , 2017). Together these findings suggest that Fab1 (PIKfyve) is dynamically and tightly regulated.…”

Section: Phosphatidylinositol 35-bisphosphate (Pi(35)p2)mentioning

confidence: 99%

PI5P and PI(3,5)P<sub>2</sub>: Minor, but Essential Phosphoinositides

Hasegawa

Strunk

Weisman

2017

Cell Struct. Funct.

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120

118

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In most eukaryotes, phosphoinositides (PIs) have crucial roles in multiple cellular functions. Although the cellular levels of phosphatidylinositol 5-phosphate (PI5P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) are extremely low relative to some other PIs, emerging evidence demonstrates that both lipids are crucial for the endocytic pathway, intracellular signaling, and adaptation to stress. Mutations that causes defects in the biosynthesis of PI5P and PI(3,5)P2 are linked to human diseases including neurodegenerative disorders. Here, we review recent findings on cellular roles of PI5P and PI(3,5)P2, as well as the pathophysiological importance of these lipids.

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Section: Phosphatidylinositol 35-bisphosphate (Pi(35)p2)mentioning

confidence: 99%

PI5P and PI(3,5)P<sub>2</sub>: Minor, but Essential Phosphoinositides

Hasegawa

Strunk

Weisman

2017

Cell Struct. Funct.

Self Cite

120

118

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“…To verify that an excess of PI(3,5)P2 inhibits hemifusion, we used vacuoles harvested from yeast expressing Fab1 with the hyperactive kinase mutation T2250A (Lang et al, 2017). As predicted, vacuoles expressing fab1 T2250A failed to undergo lipid mixing ( Fig.…”

Section: Fab1 Mutants Affect Hemifusionmentioning

confidence: 74%

“…Similarly, FAB1 was deleted by recombination using the primers 5'-FAB1-KO (5'-AGGTAGCTTCCATCCTGTACATGCAAGACCGTCACACAGCCGGATCCCCGGGTTAATTAA-3') and 3'-FAB1-KO (5'-TAAAAAAAAGTTACAGAATATAACTTGTACACGTTTATGTGAATTCGAGCTCGTTTAAAC-3') to generate BJ3505 fab1D::kanMX6 (RFY76). RFY76 was transformed with a plasmid encoding the hyperactive kinase mutant fab1 T2250A (pRS416-FAB1-T2250A) and grown in selective media lacking uracil to make RFY78 (Lang et al, 2017). Similarly, a plasmid encoding the kinase dead mutant fab1 EEE (pRS416-FAB1-EEE) was transformed into RFY76 to make RFY80 (Li et al, 2014).…”

Section: Strainsmentioning

confidence: 99%

Phosphatidylinositol 3,5-Bisphosphate Regulates Yeast Vacuole Fusion at the Transition betweentrans-SNARE Complex Formation and Hemifusion

et al. 2018

Preprint

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Phosphoinositides (PIs) regulate myriad cellular functions including membrane fusion, as exemplified by the yeast vacuole, which uses various PIs at different stages of fusion. In light of this, the effect of phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] on vacuole fusion remains unknown. PI(3,5)P2 is made by the PI3P 5-kinase Fab1/PIKfyve and has been characterized as a regulator of vacuole fission during hyperosmotic shock where it interacts with the TRP family Ca 2+ channel Yvc1. Here we demonstrate that exogenously added dioctanoyl (C8) PI(3,5)P2 abolishes homotypic vacuole fusion. This effect was not linked to interactions with Yvc1, as fusion was equally affected using yvc1D vacuoles. Thus, the effects of C8-PI(3,5)P2 on fusion versus fission operate through distinct mechanisms. Further testing showed that C8-PI(3,5)P2 inhibited vacuole fusion after the formation of trans-SNARE pairs. Although SNARE complex formation was unaffected we found that C8-PI(3,5)P2 strongly inhibited hemifusion. Overproduction of endogenous PI(3,5)P2 by the fab1 T2250A hyperactive kinase mutant also inhibited at the hemifusion stage, bolstering the model in which PI(3,5)P2 inhibits fusion when present elevated levels. Taken together, this work identifies a novel function for PI(3,5)P2 as a negative regulator of vacuolar fusion. Moreover, it suggests that this lipid acts as a molecular switch between fission and fusion.

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“…Based on these findings we hypothesized that PI(3,5)P2 may affect V-ATPase activity through modulating Ca 2+ efflux from the vacuole lumen. To start, we performed acridine orange (AO) fluorescence quenching assays with isolated vacuoles from wild type yeast as well as strains expressing the kinase deficient fab1 EEE or the hyperactive fab1 T2250A mutations (Lang et al, 2017, Li et al, 2014. Because AO fluorescence decreases in acidic environments, it can serve as a measure of V-ATPase activity and pumping of H + into the vacuole lumen.…”

Section: Proton Influx Is Modulated By Pi(35)p2mentioning

confidence: 99%

Reciprocal regulation of vacuolar calcium transport and V-ATPase activity, and the effects of Phosphatidylinositol 3,5-bisphosphate

Miner

Rivera‐Kohr

Zhang

et al. 2020

Preprint

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StatementHere we show that Ca 2+ and H + transport across the vacuole membrane is reciprocally regulated and that it is linked to the production of Phosphatidylinositol 3,5-bisphoshpate. ABSTRACTYeast vacuoles are acidified by the V-ATPase, a protein complex comprised of the membrane embedded VO complex and the soluble cytoplasmic V1 complex. The assembly of the V1-VO holoenzyme is required for the transfer of H + into the vacuole lumen for acidification. The assembly of the V1-VO holoenzyme is stabilized by the lipid phosphatidylinositol 3,5-bisphospate (PI(3,5)P2) made by the PI3P 5-kinase Fab1/PIKfyve. The absence of PI(3,5)P2 leads to the dissociation of the V1 complex from the membrane. Separately, PI(3,5)P2 has been shown to modulate Ca 2+ transport across the vacuole membrane during fission and fusion. Here we examined whether the regulation of H + and Ca 2+ by PI(3,5)P2 are interdependent. We show that modulating extraluminal Ca 2+ concentrations inhibit V-ATPase activity. As extraluminal CaCl2 levels are raised, the activity of H + pumping is reduced. Conversely, chelating free Ca 2+ with EGTA stimulated vacuole acidification. Not only did Ca 2+ levels affect H + translocation, we also show that blocking V-ATPase activity inhibited Ca 2+ transport into the vacuole lumen. Together, these data illustrate that Ca 2+ transport and V-ATPase regulation are interconnected through the modulation of vacuolar lipid profiles. Abbreviations: AO, acridine orange; DAG, diacylglycerol; FCCP, Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; PA, phosphatidic acid; PI, phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI(3,5)P2, phosphatidylinositol 3,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TRP, transient receptor potential; YPD, yeast extract, peptone, dextrose. Miner et al. -Reciprocal regulation of V-ATPase and Ca 2+ transport

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An intramolecular interaction within the lipid kinase Fab1 regulates cellular phosphatidylinositol 3,5-bisphosphate lipid levels

Cited by 16 publications

References 34 publications

PI5P and PI(3,5)P<sub>2</sub>: Minor, but Essential Phosphoinositides

PI5P and PI(3,5)P<sub>2</sub>: Minor, but Essential Phosphoinositides

Phosphatidylinositol 3,5-Bisphosphate Regulates Yeast Vacuole Fusion at the Transition betweentrans-SNARE Complex Formation and Hemifusion

Reciprocal regulation of vacuolar calcium transport and V-ATPase activity, and the effects of Phosphatidylinositol 3,5-bisphosphate

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