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J. Biol. Chem., Vol. 277, Issue 48, 46470-46477, November 29, 2002
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From the Department of Biochemistry and Molecular Biology, Medical
University of South Carolina, Charleston, South Carolina 29425
Yeast ISC1 (Yer019w) encodes
inositolphosphosphingolipid-phospholipase C and is activated by
phosphatidylserine (PS) and cardiolipin (CL) (Sawai, H., Okamoto, Y.,
Lubert, C., Mao, C., Bielawska, A., Domae, M., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 39793-39798). In
this study, the structural requirements for anionic
phospholipid-selective binding of ISC1 were determined
using site-directed and deletion mutants. FLAG-tagged Isc1p was
activated by PS, CL, and phosphatidylglycerol (PG) in a
dose-dependent manner. Using lipid-protein overlay assays, Isc1p interacted specifically and directly with PS/CL/PG.
Lipid-protein binding studies of a series of deletion mutants
demonstrated that the second transmembrane domain (TMII) and the C
terminus were required for PS binding. Moreover, the TMII and
the C terminus domain were sufficient to impart PS binding to a
heterologous protein, green fluorescence protein. In addition,
mutations of positively charged amino acid residues at the C terminus
of ISC1 reduced the activating effects of PS, suggesting
involvement of these amino acids in interaction with PS/CL/PG and in
the activation of the enzyme. Finally, when separate fragments
containing the N terminus-TMI and TMII-C terminus were expressed
heterologously, enzyme activity was reconstituted, demonstrating that
the interaction of the N terminus and the C terminus is required for
activity of Isc1p. These results raise the hypothesis that in the
presence of PS/CL/PG, the catalytic domain in the N terminus of Isc1p
is "pulled" to the membrane to interact with substrate.
These studies provide unique insights into the properties of
ISC1 and define a novel mechanism for activation of enzymes
by lipids cofactors.
Structural Requirements for Selective Binding of ISC1
to Anionic Phospholipids*
,
*
This work was supported in part by National Institutes of
Health Grant GM43825 (to Y. A. H.).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.
Recipient of Merck Company Foundation and Banyu Fellowship Awards
in Lipid Metabolism and Atherosclerosis.
§
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Medical University of South Carolina, 173 Ashley
Ave., Charleston, SC 29425. Tel.: 843-792-4321; Fax: 843-792-4322;
E-mail: hannun@musc.edu.
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