Knockdown of human Oxa1l impairs the biogenesis of F1Fo-ATP synthase and NADH:ubiquinone oxidoreductase.
The Oxa1 protein is a founding member of the evolutionarily conserved Oxa1/Alb3/YidC protein family, which is involved in the biogenesis of membrane proteins in mitochondria, chloroplasts and bacteria. The predicted human homologue, Oxa1l, was originally identified by partial functional complementation of the respiratory growth defect of the yeast oxa1 mutant. Here we demonstrate that both the endogenous human Oxa1l, with an apparent molecular mass of 42 kDa, and the Oxa1l-FLAG chimeric protein localize exclusively to mitochondria in HEK293 cells. Furthermore, human Oxa1l was found to be an integral membrane protein, and, using two-dimensional blue native/denaturing PAGE, the majority of the protein was identified as part of a 600-700 kDa complex. The stable short hairpin (sh)RNA-mediated knockdown of Oxa1l in HEK293 cells resulted in markedly decreased steady-state levels and ATP hydrolytic activity of the F(1)F(o)-ATP synthase and moderately reduced levels and activity of NADH:ubiquinone oxidoreductase (complex I). However, no significant accumulation of corresponding sub-complexes could be detected on blue native immunoblots. Intriguingly, the achieved depletion of Oxa1l protein did not adversely affect the assembly or activity of cytochrome c oxidase or the cytochrome bc(1) complex. Taken together, our results indicate that human Oxa1l represents a mitochondrial integral membrane protein required for the correct biogenesis of F(1)F(o)-ATP synthase and NADH:ubiquinone oxidoreductase.
Stiburek
Lukas
L
Department of Pediatrics and Center of Applied Genomics, 1st Faculty of Medicine, Charles University, Prague, 128 08, Czech Republic.
Fornuskova
Daniela
D
Wenchich
Laszlo
L
Pejznochova
Martina
M
Hansikova
Hana
H
Zeman
Jiri
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002645:Chickens; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D014450:Electron Transport Complex III / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005455:Fluorescent Antibody Technique; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology; D047468:Immunoprecipitation; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D013347:Subcellular Fractions
D007074:Immunoglobulin G; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; C090858:OXA1 protein; D034741:RNA, Small Interfering; D000255:Adenosine Triphosphate; D003576:Electron Transport Complex IV; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III