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J Biol Chem. 2015 Apr 10;290(15):9919-28. doi: 10.1074/jbc.M114.613984. Epub 2015 Feb 20.

The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

Author information

1
From the Department of Chemistry and Biology and the Molecular Science Program, Ryerson University, Toronto, Ontario M5B2K3, Canada and.
2
From the Department of Chemistry and Biology and.
3
the Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G1X8, Canada.
4
From the Department of Chemistry and Biology and the Molecular Science Program, Ryerson University, Toronto, Ontario M5B2K3, Canada and the Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G1X8, Canada rbotelho@ryerson.ca.

Abstract

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes.

KEYWORDS:

Lysosomal Acidification; Lysosome; Organellar pH Homeostasis; Organelle; Phosphoinositide; Yeast

PMID:
25713145
PMCID:
PMC4392288
DOI:
10.1074/jbc.M114.613984
[Indexed for MEDLINE]
Free PMC Article

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