Abstract
The low-level endo-lysosomal signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), is required for full assembly and activity of vacuolar H+-ATPases (V-ATPases) containing the vacuolar a-subunit isoform Vph1 in yeast. The cytosolic N-terminal domain of Vph1 is also recruited to membranes in vivo in a PI(3,5)P2-dependent manner, but it is not known if its interaction with PI(3,5)P2 is direct. Here, using biochemical characterization of isolated yeast vacuolar vesicles, we demonstrate that addition of exogenous short-chain PI(3,5)P2 to Vph1-containing vacuolar vesicles activates V-ATPase activity and proton pumping. Modeling of the cytosolic N-terminal domain of Vph1 identified two membrane-oriented sequences that contain clustered basic amino acids. Substitutions in one of these sequences (231KTREYKHK) abolished the PI(3,5)P2-dependent activation of V-ATPase without affecting basal V-ATPase activity. We also observed that vph1 mutants lacking PI(3,5)P2 activation have enlarged vacuoles relative to those in WT cells. These mutants exhibit a significant synthetic growth defect when combined with deletion of Hog1, a kinase important for signaling the transcriptional response to osmotic stress. The results suggest that PI(3,5)P2 interacts directly with Vph1, and that this interaction both activates V-ATPase activity and protects cells from stress.
Highlights
The low-level endo-lysosomal signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI[3,5]P2), is required for full assembly and activity of vacuolar H؉-ATPases (V-ATPases) containing the vacuolar a-subunit isoform Vph1 in yeast
Keeping in mind increasing evidence of involvement of basic and aromatic amino acids in noncanonical Phosphatidylinositol phosphate (PIP) and PI[3,5]P2 binding, we looked for amino acid patches with these characteristics in regions of Vph1-N-terminal domain (NT) facing the organelle membrane (Fig. 2A) [40, 41]
The vph1-D1 and -D2 mutants were able to establish a MgATP-driven proton gradient as indicated by an initial fluorescent quench, PI[3,5]P2 failed to give any significant additional quenching (Fig. 3C). These results indicate that ATPdriven proton pumping is enhanced by PI[3,5]P2 and compromised by mutations that prevented ATPase activation
Summary
Exogenous PI[3,5]P2 enhances the activity of V-ATPases in isolated vacuolar vesicles. The vph1-D1 and -D2 mutants were able to establish a MgATP-driven proton gradient as indicated by an initial fluorescent quench, PI[3,5]P2 failed to give any significant additional quenching (Fig. 3C) These results indicate that ATPdriven proton pumping is enhanced by PI[3,5]P2 and compromised by mutations that prevented ATPase activation. The vph1-D1 and vph1-D2 mutants exhibit an intermediate phenotype, maintaining a significantly larger vacuolar diameter than WT cells, but not as large as vac14⌬ cells (Fig. 4, B and C) In combination, these results indicate that V-ATPase activation by PI[3,5]P2 may contribute to maintaining vacuolar size and morphology under normal and hyperosmotic conditions but does not fully account for the phenotypes seen when PI[3,5]P2 is missing. Our result suggests that failure of PI[3,5]P2-dependent V-ATPase activation can be tolerated, even in the presence of 0.5 M NaCl, but a synthetic growth defect is uncovered upon loss of stress response pathways like the HOG pathway
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