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Structural requirements for selective binding of ISC1 to anionic phospholipids
Yasuo Okamoto1, Silvia Vaena De Avalos, Yusuf A Hannun
1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
The Journal of Biological Chemistry
|September 24, 2002
Summary
Yeast ISC1 enzyme requires specific lipid interactions for activation. The second transmembrane domain and C-terminus are crucial for binding anionic phospholipids like phosphatidylserine, enabling enzyme activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Yeast ISC1 (Yer019w) encodes inositolphosphosphingolipid-phospholipase C.
- ISC1 activation is known to be influenced by anionic phospholipids such as phosphatidylserine (PS) and cardiolipin (CL).
Purpose of the Study:
- To determine the structural requirements for anionic phospholipid-selective binding of ISC1.
- To elucidate the mechanism by which lipids activate ISC1 enzyme activity.
Main Methods:
- Site-directed and deletion mutagenesis of ISC1.
- Lipid-protein overlay assays and lipid-protein binding studies.
- Heterologous expression of ISC1 fragments and fusion proteins.
Main Results:
- ISC1 specifically and directly interacts with PS, CL, and phosphatidylglycerol (PG).
- The second transmembrane domain (TMII) and C-terminus of ISC1 are essential for PS binding and sufficient to confer PS binding to a heterologous protein.
- Mutations in C-terminal positively charged residues diminish PS activation, implicating them in lipid interaction and enzyme activation.
- Reconstitution of enzyme activity from N-terminus-TMI and TMII-C-terminus fragments indicates the necessity of N- and C-terminus interaction.
Conclusions:
- The TMII and C-terminus of ISC1 mediate direct and specific binding to anionic phospholipids.
- Positively charged residues at the C-terminus are involved in the interaction with PS, CL, and PG.
- A novel mechanism for enzyme activation by lipid cofactors is proposed, where anionic phospholipids recruit the catalytic N-terminus to the membrane via interactions mediated by the TMII and C-terminus.