Regulation of vacuolar acidity by PtdIns3P and PtdIns(35)P2 in Saccharomyces cerevisiae.

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Yeasts are single celled organisms and contain an internal compartment called the vacuole, which digests unwanted proteins (a bit like the stomach) and stores food. For proper function the inside of the vacuole must be made acidic (again like the stomach), and this is mediated by a tiny pump called the vacuolar ATPase (V-ATPase). The V-ATPase is on the surface of the vacuole and pumps protons (acidity is caused by an excess of protons) into the vacuole interior in a process that requires considerable expenditure of energy. The vacuole is encapsulated by a membrane consisting of lipids, which separates it from the rest of the cell. We are particularly interested in two lipids, which are part of a group called polyphosphoinositides (PPIn). These two PPIn, although comprising only a very small fraction of the lipids in the vacuole membrane, are responsible for mediating a wide variety of functions and are required for the vacuole to be come acidic. We are trying to find out how these PPIn are able to change the acidity of the interior of the vacuole. Of particular interest will be how the PPIns might affect the V-ATPase pump. An example of the type of experiments that we will do is to purifiy vacuoles from yeast and add PPIn to them; we can then measure how active the V-ATPase pump is in the presence of different PPIn. By doing such experiments we should establish a mechanistic link between PPIn formation and vacuole acidity. Why is the research important? Firstly, yeasts are responsible for contamination and spoilage of a significant proportion of the World's food supply. Weak acids are frequently used food additives that have anti-fungal action. Addition of such weak acids to yeast causes changes in cellular PPIn levels and intracellular pH. Our work will investigate PPIn function and vacuolar acidification, and allow greater understanding of yeast stress responses. Ultimately, knowledge of yeast stress responses will lead to the design of better anti-fungal food additives. Additionally it is likely that the mechanisms of pH regulation by PPIn will be shared other organisms that have a vacuole such as plants. Plants, like yeasts, have to cope with a variety of stresses, so our work may well have wide implications for agriculture.