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.
Nijmegen Center for Mitochondrial Disorders, Department of Pediatrics, University Medical Center Nijmegen, Geert Grooteplein 10, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
Cristina
C
Ugalde
Rutger
R
Vogel
Richard
R
Huijbens
Bert
B
Van Den Heuvel
Jan
J
Smeitink
Leo
L
Nijtmans
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