Subunits of mitochondrial complex I exist as part of matrix- and membrane-associated subcomplexes in living cells.
Mitochondrial complex I (CI) is a large assembly of 45 different subunits, and defects in its biogenesis are the most frequent cause of mitochondrial disorders. In vitro evidence suggests a stepwise assembly process involving pre-assembled modules. However, whether these modules also exist in vivo is as yet unresolved. To answer this question, we here applied submitochondrial fluorescence recovery after photobleaching to HEK293 cells expressing 6 GFP-tagged subunits selected on the basis of current CI assembly models. We established that each subunit was partially present in a virtually immobile fraction, possibly representing the holo-enzyme. Four subunits (NDUFV1, NDUFV2, NDUFA2, and NDUFA12) were also present as highly mobile matrix-soluble monomers, whereas, in sharp contrast, the other two subunits (NDUFB6 and NDUFS3) were additionally present in a slowly mobile fraction. In the case of the integral membrane protein NDUFB6, this fraction most likely represented one or more membrane-bound subassemblies, whereas biochemical evidence suggested that for the NDUFS3 protein this fraction most probably corresponded to a matrix-soluble subassembly. Our results provide first time evidence for the existence of CI subassemblies in mitochondria of living cells.
Dieteren
Cindy E J
CE
Department of Biochemistry, Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Willems
Peter H G M
PH
Vogel
Rutger O
RO
Swarts
Herman G
HG
Fransen
Jack
J
Roepman
Ronald
R
Crienen
Gijs
G
Smeitink
Jan A M
JA
Nijtmans
Leo G J
LG
Koopman
Werner J H
WJ
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D042967:Electron Transport Complex I / Q000737:chemistry / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry; D009245:NADH Dehydrogenase / Q000737:chemistry; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013997:Time Factors
D018076:DNA, Complementary; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009247:NADH, NADPH Oxidoreductases; D009245:NADH Dehydrogenase; D042967:Electron Transport Complex I; C527690:NDUFB6 protein, human; C472512:NDUFS3 protein, human