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(Received for publication, July 1, 1996, and in revised form, December 30, 1996)
From the Department of Biochemistry and the Lucille P. Markey
Cancer Center, University of Kentucky Medical Center,
Lexington, Kentucky 40536-0084
We have identified a Saccharomyces
cerevisiae gene necessary for the step in sphingolipid synthesis
in which inositol phosphate is added to ceramide to form
inositol-P-ceramide, a reaction catalyzed by
phosphatidylinositol:ceramide phosphoinositol transferase (IPC synthase). This step should be an effective target for antifungal drugs. A key element in our experiments was the development of a
procedure for isolating mutants defective in steps in sphingolipid synthesis downstream from the first step including a mutant defective in IPC synthase. An IPC synthase defect is supported by data showing a
failure of the mutant strain to incorporate radioactive inositol or
N-acetylsphinganine into sphingolipids and, by using an
improved assay, a demonstration that the mutant strain lacks enzyme
activity. Furthermore, the mutant accumulates ceramide when fed
exogenous phytosphingosine as expected for a strain lacking IPC
synthase activity. Ceramide accumulation is accompanied by cell death, suggesting the presence of a ceramide-activated death response in
yeast. A gene, AUR1 (YKL004w), that complements the IPC
synthase defect and restores enzyme activity and sphingolipid synthesis was isolated. Mutations in AUR1 had been shown previously
to give resistance to the antifungal drug aureobasidin A, leading us to predict that the drug should inhibit IPC synthase activity. Our data
show that the drug is a potent inhibitor of IPC synthase with an
IC50 of about 0.2 nM. Fungal pathogens are an
increasing threat to human health. Now that IPC synthase has been shown
to be the target for aureobasidin A, it should be possible to develop high throughput screens to identify new inhibitors of IPC synthase to
combat fungal diseases.
Volume 272, Number 15,
Issue of April 11, 1997
pp. 9809-9817
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
COMPLEMENTATION OF THE INOSITOL PHOSPHORYLCERAMIDE SYNTHASE
DEFECT IN A MUTANT STRAIN OF SACCHAROMYCES CEREVISIAE BY THE
AUR1 GENE
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M. S. Skrzypek, M. M. Nagiec, R. L. Lester, and R. C. Dickson Inhibition of Amino Acid Transport by Sphingoid Long Chain Bases in Saccharomyces cerevisiae J. Biol. Chem., January 30, 1998; 273(5): 2829 - 2834. [Abstract] [Full Text] [PDF]
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