Human mitochondrial complex I assembles through the combination of evolutionary conserved modules: a framework to interpret complex I deficiencies.
With 46 subunits, human mitochondrial complex I is the largest enzyme of the oxidative phosphorylation system. We have studied the assembly of complex I in cultured human cells. This will provide essential information about the nature of complex I deficiencies and will enhance our understanding of mitochondrial disease mechanisms. We have found that 143B206 rho zero cells, not containing mitochondrial DNA, are still able to form complex I subcomplexes. To further address the nature of these subcomplexes, we depleted 143B osteosarcoma cells of complex I by inhibiting mitochondrial protein translation with doxycycline. After removing this drug, complex I formation resumes and assembly intermediates were observed by two-dimensional blue native electrophoresis. Analysis of the observed subcomplexes indicates that assembly of human complex I is a semi-sequential process in which different preassembled subcomplexes are joined to form a fully assembled complex. The membrane part of the complex is formed in distinct steps. The B17 subunit is part of a subcomplex to which ND1, ND6 and PSST are subsequently added. This is bound to a hydrophilic subcomplex containing the 30 and 49 kDa subunits, to which a subcomplex including the 39 kDa subunit is incorporated, and later on the 18 and 24 kDa subunits. At a later stage more subunits, including the 15 kDa, are added and holo-complex I is formed. Our results suggest that human complex I assembly resembles that of Neurospora crassa, in which a membrane arm is formed and assembled to a preformed peripheral arm, and support ideas about modular evolution.
Ugalde
Cristina
C
Nijmegen Center for Mitochondrial Disorders, Department of Pediatrics, University Medical Center Nijmegen, Geert Grooteplein 10, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
Vogel
Rutger
R
Huijbens
Richard
R
Van Den Heuvel
Bert
B
Smeitink
Jan
J
Nijtmans
Leo
L
D004272:DNA, Mitochondrial / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D042967:Electron Transport Complex I / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D028361:Mitochondrial Diseases / Q000201:enzymology* / Q000235:genetics; D008954:Models, Biological; D012516:Osteosarcoma; D021381:Protein Transport / Q000187:drug effects; D014407:Tumor Cells, Cultured
D004272:DNA, Mitochondrial; D042967:Electron Transport Complex I; D004318:Doxycycline