Differential Partitioning of Lipids Metabolized by Separate
Yeast Glycerol-3-phosphate Acyltransferases Reveals That Phospholipase
D Generation of Phosphatidic Acid Mediates Sensitivity to
Choline-containing Lysolipids and Drugs*
Vanina
Zaremberg and
Christopher R.
McMaster
From the Atlantic Research Centre, Departments of Pediatrics and
Biochemistry and Molecular Biology, IWK Health Centre, Dalhousie
University, Halifax, Nova Scotia B3H 4H7, Canada
In this study we demonstrate that the
GAT1 and GAT2 genes encode the major
glycerol-3-phosphate acyltransferase activities in Saccharomyces
cerevisiae. Genetic inactivation of either GAT1 or
GAT2 did not alter cell growth but inactivation of both
resulted in growth cessation. Metabolic analyses of gat1
and gat2 yeast detected that the major differences were:
(i) a 50% increase in the rate of triacylglycerol synthesis in
gat1 yeast and a corresponding 50% decrease in
gat2 yeast, and (ii) a 5-fold increase in
glycerophosphocholine production through deacylation of
phosphatidylcholine synthesized through the CDP-choline pathway in
gat1 yeast, whereas gat2 yeast displayed a
10-fold decrease. To address why we observed alterations in
phospholipid turnover specific to phosphatidylcholine produced through
the CDP-choline pathway in gat1 and gat2 yeast
we tested their sensitivity to various cytotoxic lysolipids and
observed that gat2 cells were more sensitive to
lysophosphatidylcholine, but not other lysolipids. To pursue the
mechanism we analyzed their sensitivity to choline-containing
lysolipids or drugs that could not be deacylated and/or reacylated. Our
data showed that gat1 and gat2 yeast were
resistant and sensitive to lysoplatelet activating factor, platelet
activating factor, and the anti-tumor lipid edelfosine, respectively,
indicating that their sensitivity to these compounds was not because of
differences in rates of phosphatidylcholine deacylation. As
growth of gat2 cells was impaired in the presence of
ethanol, a phospholipase D (Spo14p) inhibitor, we inferred that
phospholipase D may play important biologic and metabolic roles in
phenotypes observed in gat yeast. Genetic inactivation of
the SPO14 gene resulted in increased susceptibility,
whereas expression of Escherichia coli diacylglycerol
kinase relieved growth inhibition, to choline-containing lysolipids and
drugs. Our results are consistent with a model whereby phosphatidic
acid generated from phosphatidylcholine hydrolysis by Spo14p regulates susceptibility to choline-containing lysolipid analogs and drugs.
*
This work was supported by individual and group operating
grants from the Canadian Institutes of Health Research and an
IWK Health Centre Senior Research Scholar award (to C. R. M.), as well as a postdoctoral fellowship from Cancer Care Nova
Scotia (to V. Z.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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