Loss of regulators of V-ATPase function and ceramide synthesis results in multi-drug sensitivity in S. pombe

Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK

^* To whom correspondence should be addressed. Email: njones{at}picr.man.ac.uk. cwilkinson{at}picr.man.ac.uk.

	Abstract

We undertook a screen to isolate determinants of drug resistance in fission yeast and identified two genes, which when mutated result in sensitivity to a range of structurally unrelated compounds, some of them commonly used in the clinic. One gene, rav1, encodes the homologue of a budding yeast protein which regulates the assembly of the vacuolar ATPase. The second gene, lac1, encodes a homologue of genes that are required for ceramide synthesis. Both mutants are sensitive to the chemotherapeutic agent doxorubicin and using the naturally fluorescent properties of this compound, we found that both rav1 and lac1 mutations result in an increased accumulation of the drug in cells. The multi-drug sensitive phenotype of rav1 mutants can be rescued by up-regulation of the lag1 gene which encodes a homologue of lac1 whereas overexpression of either lac1 or lag1 confers multi-drug resistance to wild type cells. These data suggest that changing the amount of ceramide synthase activity in cells can influence innate drug resistance.

The function of Rav1 appears to be conserved as we show that SpRav1 is part of a RAVE-like complex in fission yeast and that loss of rav1 results in defects in V-ATPase activity. Thus we conclude that loss of normal V-ATPase function results in an increased sensitivity of S. pombe cells to drugs.

The rav1 and lac1 genes are conserved in both higher eukaryotes and various pathogenic fungi. Thus our data could provide the basis of strategies to sensitize tumour cells or drug resistant pathogenic fungi to drugs.