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J. Biol. Chem., Vol. 279, Issue 16, 15968-15974, April 16, 2004

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Docking of Endothelial Nitric Oxide Synthase (eNOS) to the Mitochondrial Outer Membrane

A PENTABASIC AMINO ACID SEQUENCE IN THE AUTOINHIBITORY DOMAIN OF eNOS TARGETS A PROTEINASE K-CLEAVABLE PEPTIDE ON THE CYTOPLASMIC FACE OF MITOCHONDRIA^*

Shujuan Gao, Jun Chen¶, Sergey V. Brodsky¶, Harer Huang¶, Stephen Adler¶, Juliane H. Lee||, Neetu Dhadwal¶, Leona Cohen-Gould||, Steven S. Gross||, and Michael S. Goligorsky¶**

From the Department of Pharmacology, State University of New York, Stony Brook, New York 11794, ^¶Departments of Medicine and Pharmacology, New York Medical College, Valhalla, New York 10595, and ^||Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021

Despite growing evidence for a mitochondrial localization of nitric oxide (NO) synthase and a broadening spectrum of NO actions on mitochondrial respiration and apoptosis, the basis for interaction between the enzyme and the organelle remain obscure. Here we investigated mitochondrial localization of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells and human embryonic kidney cells transfected or infected with eNOS expression vectors. Copurification of eNOS with mitochondria was observed in both human umbilical vein endothelial cells and eNOS-expressing human embryonic kidney cells. Immunodetectable eNOS was cleaved from mitochondria by proteinase K treatment, suggesting eNOS association with the outer mitochondrial membrane. Localization of eNOS to a proteinase K-cleavable site on the cytoplasmic face of the outer membrane was confirmed by immunogold labeling of non-permeabilized mitochondria. Markers for mitochondrial subfractions ruled out the possibility of eNOS association with an intramitochondrial site or inverted mitochondrial particles. Denaturation of eNOS did not attenuate association with mitochondria. Mutant eNOS lacking a pentabasic amino acid sequence within the autoinhibitory domain (residues 628–632 of the bovine eNOS) showed dramatically reduced binding to the mitochondrial but not to the plasma membrane, which was associated with increased oxygen consumption. Collectively, these findings argue in favor of eNOS localization to the outer mitochondrial membrane in endothelial cells and identify elements of a novel anchoring mechanism.

Received for publication, August 4, 2003 , and in revised form, January 23, 2004.

^* This work was supported in part by National Institutes of Health Grants DK45462, DK54602, and DK52783 (to M. S. G.). 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.

To whom correspondence should be addressed: Dept. of Pharmacology, SUNY Stony Brook, NY 11794-8152. Tel.: 631-444-8018; Fax: 631-444-7641; E-mail: gao{at}pharm.sunysb.edu. ** To whom correspondence should be addressed: Depts. of Medicine and Pharmacology, New York Medical College, Valhalla, NY 10595. Tel.: 914-594-4730; Fax: 914-594-4732; E-mail: Michael_Goligorsky{at}nymc.edu.

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