Influence of subunit transcript and protein levels on formation of a mitochondrial multienzyme complex.
Constitutive expression of nuclear genes encoding mitochondrial proteins raises the question of whether these proteins are present in similar amounts in mitochondria of different tissues. We report that amounts of a single multienzyme complex can vary on a per mitochondrion basis depending on the number of mitochondria per cell. Human branched-chain alpha-keto acid dehydrogenase (BCKD) expression is used as a paradigm in these studies. Expression is compared and contrasted in HepG2 and DG75 cells in which mitochondrial content is twofold higher in the hepatocarcinoma line than in the lymphoblastoid line. Per cell, BCKD activity is equal in the two cells types, but BCKD protein concentration per mitochondrion is twofold higher in DG75 cells. Steady-state mRNA levels do not appear to be directly related to amounts of protein in the two cell lines. To test whether one subunit is limiting in formation of complex, overexpression of each BCKD subunit was elicited by plasmid transfection of the DG75 cells. Only overexpression of the beta-subunit of the decarboxylase component induced more BCKD activity without apparent increase in mRNA for the other endogenously expressed subunits. This implies that free BCKD subunits exist in a cell and can be recruited into an active complex when the limiting subunit becomes available.
McConnell
B B
BB
Department of Genetics and Molecular Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
McKean
M C
MC
Danner
D J
DJ
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D015139:Blotting, Southern; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000201:enzymology; D018929:Cell Culture Techniques; D005434:Flow Cytometry; D006207:Half-Life; D006801:Humans; D007658:Ketone Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics*; D010957:Plasmids; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000201:enzymology; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000032:analysis; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D009097:Multienzyme Complexes; D012333:RNA, Messenger; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)