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Science 329 (5995): 1085-1088

Copyright © 2010 by the American Association for the Advancement of Science

Phosphatidic Acid Is a pH Biosensor That Links Membrane Biogenesis to Metabolism

Barry P. Young1,*, John J. H. Shin1,*, Rick Orij2, Jesse T. Chao1, Shu Chen Li1, Xue Li Guan3,4, Anthony Khong5, Eric Jan5, Markus R. Wenk4,6,7, William A. Prinz8, Gertien J. Smits2, and Christopher J. R. Loewen1,9,{dagger}

1 Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
2 Department of Molecular Biology and Microbial Food Safety, University of Amsterdam, Amsterdam, 1018 WV, Netherlands.
3 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119077.
4 Department of Biochemistry, University of Geneva, Sciences II, 30 Quai Ernest Ansermet, CH-1211 Geneva, Switzerland.
5 Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
6 Department of Biological Sciences National University of Singapore, Singapore 119077.
7 Swiss Tropical and Public Health Institute, University of Basel, Socinstrasse 57, P.O. Box 4002, Basel, Switzerland.
8 Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
9 The Brain Research Centre, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.


Figure 1
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  		Fig. 1. Genome-wide screen for regulators of phospholipid metabolism. (A) Inositol auxotrophy of ~4800 deletion mutants and effect of deletion of OPI1. Plotted are log2 values of ratios of colony sizes for growth of mutants in the absence or presence of inositol (5). Single mutants are plotted on the x axis and double mutants with {triangleup}opi1 on the y axis. (B) Inositol auxotrophy of known regulators of phospholipid metabolism and rescue by {triangleup}opi1. Plotted are ratios of colony sizes for growth of mutants in the absence (–Ino) or presence (+Ino) of inositol. (C) Inositol auxotrophy of V-ATPase deletion mutants. Mutants are grouped by V-ATPase domain (V1, peripherally associated subunits; V0, membrane-associated subunits) or factors required for V-ATPase assembly. Genes in parentheses indicate deletion of an overlapping dubious open reading frame and may not be true nulls. (D) Inositol auxotrophy of pma1-007 and {triangleup}trk1 mutants. Error bars indicate SD.

 

Figure 2
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  		Fig. 2. pH regulates phospholipid metabolism. (A) pHi of mutants grown in medium at pH 3, 4, and 5 compared to the wild type (WT) (*, versus WT at a given pH, P < 0.001). (B) UASINO reporter expression measured in different mutants grown at pH 3, 4, and 5 (*, versus pH 5, P < 0.001). (C) Growth of mutants in the absence of inositol at varying pH at 37°C. (D) Nuclear localization of GFP-Opi1 in cells grown at pH 3 and 5 quantified by confocal microscopy (*, versus WT at a given pH, P < 0.005; **, versus pma1-007 at pH 5, P < 0.01). (E) Effect on pHi after addition of 100 µM ebselen to WT and pma1-007 cells grown in medium at pH 5 (*, versus WT at a given time point, P < 0.05). (F) Effect on the localization of GFP-Opi1 5 min after addition of 100 µM ebselen (+ebs). Arrows indicate ER localizations (straight, cortical; jagged, nuclear envelope); arrowheads indicate cytoplasmic (straight) and nuclear (jagged) localizations. Error bars indicate SEM in (A), (D), and (E) and SD (B). Scale bars, 2 µm.

 

Figure 3
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  		Fig. 3. pH governs the binding of Opi1 to PA through its protonation state. (A) Localization of GFP-Q2 after 5 min of ebselen treatment. (B) Localization of the PA-binding domain of Spo20 (GFP-Spo2061-91) after 5 min of ebselen treatment. (C) Treatment of yeast expressing GFP-Q2 with CCCP buffered at the indicated pH. (D) Quantification of PM localization of GFP-Q2 with CCCP treatment (*, versus pH 6.4; **, versus pH 6.8; ***, versus pH 7; P < 0.005). (E) GFP-Q2 localization in {triangleup}vma2 cells. (F) pHi measured in WT and {triangleup}vma2 cells grown in pH 5 medium (*P < 0.0001). (G) Total PA measured by mass spectrometry in WT and {triangleup}vma2 cells grown in pH 5 medium (*P < 0.0001). (H) Binding of Q2 and Q2C3M to liposomes containing 10 mol % PA, 40 mol % phosphatidylethanolamine (PE) over a range of pH values (*, versus pH 6.4; **, versus pH 6.8; ***, versus pH 7.2; P < 0.05). (I) Binding of Q2 to liposomes (0, 100, 200 µM total lipid) containing 50 mol % PA or methyl-PA at pH 7.2. (J) Binding of Q2 and Q2C3M to liposomes containing 20 mol % methyl-PA, 40 mol % PE over a range of pH values. Error bars indicate SD except in (D) (SEM). Scale bars, 2 µm.

 

Figure 4
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  		Fig. 4. Nutrient sensing and phospholipid metabolism are co-regulated by pH. (A) Localization of GFP-Opi1 in WT cells during glucose starvation. Time after removal from glucose (–Dex) is shown. Arrows and arrowheads as in Fig. 2. (B) Quantification of nuclear GFP-Opi1 after glucose starvation in WT and {triangleup}reg1 cells (*, versus WT at a given time point, P < 0.0001). (C) Change in pHi measured in WT and {triangleup}reg1 cells during glucose starvation (*, versus t = 0 for {triangleup}reg1, P < 0.001). (D) INO1 mRNA levels during glucose starvation measured by Northern blot (+ Ino, cells grown in medium with inositol). (E) Growth of mutants at pH 4 in the presence or absence of inositol at 37°C. (F) Pma1 specific activity measured in WT and {triangleup}reg1 cells before and 20 min after glucose starvation (*, versus WT +Dex, P < 0.05; **, versus WT +Dex, P < 0.005). Scale bar, 2 µm.

 

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