Faculty Collaboration Database

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. J Biol Chem 2004 Apr 16;279(16):15968-74

Date

02/06/2004

Pubmed ID

14761967

DOI

10.1074/jbc.M308504200

Scopus ID

2-s2.0-11144356896 (requires institutional sign-in at Scopus site)   109 Citations

Abstract

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.

Author List

Gao S, Chen J, Brodsky SV, Huang H, Adler S, Lee JH, Dhadwal N, Cohen-Gould L, Gross SS, Goligorsky MS

MESH terms used to index this publication - Major topics in bold

Biological Transport
Cell Line
Endopeptidase K
Humans
Intracellular Membranes
Microscopy, Immunoelectron
Mitochondria
Mutation
Nitric Oxide Synthase
Nitric Oxide Synthase Type III