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.
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.
Gao
Shujuan
S
Department of Pharmacology, State University of New York, Stony Brook, New York 11794, USA.
Chen
Jun
J
Brodsky
Sergey V
SV
Huang
Harer
H
Adler
Stephen
S
Lee
Juliane H
JH
Dhadwal
Neetu
N
Cohen-Gould
Leona
L
Gross
Steven S
SS
Goligorsky
Michael S
MS
D001692:Biological Transport; D002460:Cell Line; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D008928:Mitochondria / Q000201:enzymology* / Q000648:ultrastructure; D009154:Mutation; D019001:Nitric Oxide Synthase / Q000378:metabolism* / Q000648:ultrastructure; D052250:Nitric Oxide Synthase Type III
C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019286:Endopeptidase K