DESCRIPTION (Investigator's Abstract): The long range goals of this project are: (1) To understand the compartmentalization of mammalian mitochondrial precursor proteins encoded by the nuclear genome and post-translationally incorporated into mitochondria, an event mediated by NH2-terminal transit peptides. (2) To delineate structure-function relationships of one such mitochondrial protein, medium chain acyl-CoA dehydrogenase (MCAD). (3) To characterize the common cis-acting regulatory DNA elements in human nuclear genes encoding MCAD and mitochondrial malate dehydrogenase (mMDH) which control their coordinated expression during mitochondrial biogenesis. The Specific Aims include: (1) Comparison in vitro of mMDH and MCAD uptake into mitochondria of different tissues. (2) Analysis of uptake in vivo of mMDH transit peptide mutants after transfection into Cos cells. (3) Expression of MCAD and mMDH precursor proteins in sufficient quantities for structural characterization by crystallography and x-ray diffraction. (4) Isolation of the transit peptide receptor and pore proteins which are part of the translocation apparatus. (5) Delineation of the molecular basis of MCAD deficiency in human patients. (6) Analysis of a known MCAD human MCAD mutant protein after expression in mammalian cells. (7) Structural studies of mutations of essential residues in human MCAD. (8) Characterization of the human mMDH gene. (9) Analysis of cis-regulatory elements of the mMDH and MCAD genes after transfection into muscle, hepatoma, and intestinal cells. (10) Isolation of tissue-specific and developmental regulatory elements of these genes in transgenic mice. In vitro mutagenesis techniques by the polymerase chain reaction (PCR) of both transit peptides and mature proteins; precursor protein uptake in vitro into mitochondria; transfection into mammalian cells and in vivo uptake into mitochondria; expression in both bacteria and mammalian cells to generate large amounts of precursor proteins; crystallography and x-ray diffraction with computer analysis of normal, mutant, and precursor proteins for structural comparisons; PCR analysis of MCAD mRNAs to isolate natural mutants; genomic and cDNA cloning; transfection into mammalian cells of regulatory elements fused to reporter genes; and analysis of regulatory elements fused to reporter genes in transgenic animals will be used to achieve these Specific Aims. These studies will begin to delineate the basic mechanisms of mitochondrial biogenesis and characterize the molecular and structural basis for MCAD deficiency, a common cause of sudden infant death and Reye's syndrome.