Mitochondrial import and processing of an in vitro synthesized human prebranched chain acyltransferase fragment.
A 1.6-kb cDNA for human liver branched-chain acyltransferase [E2b] was placed in a transcription vector under the control of the SP6 promoter. In vitro translation of transcripts from this vector produced a pre-E2b fragment of Mr 39,000. Following import into mitochondria, this protein was processed to a protein with an Mr of 36,000. The processed protein was fully protected from trypsin digestion. Import and processing did not occur in the presence of rhodamine 123 or carbonyl cyanide m-chlorophenyl hydrazone, suggesting that membrane potential and coupled respiration were required. Uptake and processing were species and tissue independent, since both mouse-liver and human-lymphoblast mitochondria converted the human pre-E2b protein fragment. Mitochondria from patient cells that lack E2b through an inherited defect were able to import and process the in vitro-made protein, suggesting that the inherited defect was in the gene for E2b and not in the organelle-structure function. This system now provides additional methods for investigation of mechanisms responsible for the human inherited disorders affecting the branched-chain alpha-ketoacid dehydrogenase complex.
Litwer
S
S
Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322.
Danner
D J
DJ
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000217:Acyltransferases / Q000235:genetics*; D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007658:Ketone Oxidoreductases / Q000235:genetics; D008099:Liver / Q000201:enzymology; D008375:Maple Syrup Urine Disease / Q000201:enzymology / Q000235:genetics*; D051379:Mice; D008930:Mitochondria, Liver / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational
D009097:Multienzyme Complexes; D004247:DNA; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000217:Acyltransferases