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J. Biol. Chem., Vol. 281, Issue 46, 34897-34908, November 17, 2006

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The Chemistry of Phospholipid Binding by the Saccharomyces cerevisiae Phosphatidylinositol Transfer Protein Sec14p as Determined by EPR Spectroscopy^*

Tatyana I. Smirnova¹, Thomas G. Chadwick, Ryan MacArthur, Oleg Poluektov, Likai Song^¶, Margaret M. Ryan^||2, Gabriel Schaaf^||23, and Vytas A. Bankaitis^||4

From the Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, Argonne National Laboratory, Argonne, Illinois 60439, ^¶Florida State University, Tallahassee, Florida 32310, and the ^||Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7090

The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C₅ region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer.

Received for publication, March 30, 2006 , and in revised form, August 31, 2006.

^* The work at North Carolina State University was supported by National Science Foundation Grant MCB-0451510 with partial support from American Chemical Society Petroleum Research Fund Grant 40771-G4 (both to T. I. S.). The work at Argonne National Laboratory was supported by the United States Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences, under Contract W-31-109-Eng-38. The National Biomedical EPR Center is supported by National Institutes of Health (NIH) Grant EB001980. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Supported by NIH Grant GM44530 (to V. A. B.).

³ Supported by a postdoctoral training grant from the Deutsche Forschungsgemeinschaft.

¹ To whom correspondence may be addressed: Dept. of Chemistry, North Carolina State University, 2620 Yarborough Dr., Raleigh, NC 27695. Tel.: 919-513-4375; Fax: 919-513-7353; E-mail: Tatyana_Smirnova{at}ncsu.edu. ⁴ To whom correspondence may be addressed: Dept. of Cell & Developmental Biology, 108 Taylor Hall, University of North Carolina, Chapel Hill, NC 27599-7090. Tel.: 919-962-9870; Fax: 919-966-1856; E-mail: vytas{at}med.unc.edu.

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