ABSTRACT
Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. Mutations in DDP1 (deafness dystonia peptide 1), the human homolog of Tim8, are associated with the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder. We show that DDP1 acts with human Tim13 in a complex in the intermembrane space. The DDP1.hTim13 complex is in direct contact with translocation intermediates of human Tim23 in mammalian mitochondria. The human DDP1.hTim13 complex complements the function of the TIM8.13 complex in yeast and facilitates import of yeast and human Tim23. Thus, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may involve an impaired biogenesis of the human TIM23 complex causing severe pleiotropic mitochondrial dysfunction.
Subject(s)
Carrier Proteins/metabolism , DNA-Binding Proteins/physiology , Membrane Proteins/metabolism , Membrane Transport Proteins , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins , Saccharomyces cerevisiae Proteins , Animals , Biological Transport , Carrier Proteins/chemistry , Carrier Proteins/physiology , Humans , Intracellular Membranes/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , PolymersABSTRACT
The gene for human cytochrome c oxidase subunit VIIa liver isoform (COX7AL) was isolated and its sequence determined and analyzed. The three introns of the gene are considerably larger than those of the heart isoform of subunit VIIa (COX7AH), but the position of the introns relative to the cDNA sequences is homologous between the two genes. Comparison with other isolated COX7AL genes suggests that the promoter region binding motifs for transcription factors have evolved along with the coding region. In fibroblasts cultured originally from a Leigh's disease patient, a shortened COX7AL cDNA was identified by RT-PCR, consisting of exon I joined to exon IV, omitting exons II and III. No mutation could be identified in COX7AL of the patient, suggesting that the shortened cDNA is due to an alteration of the genome during cell culture. A surprising transcription of COX7AH was observed in cultured fibroblasts, suggesting a potential utility of these cells for study of its gene expression.
Subject(s)
Electron Transport Complex IV/genetics , Genome, Human , Amino Acid Sequence , Base Sequence , Cells, Cultured , DNA/analysis , Electron Transport Complex IV/isolation & purification , Fibroblasts/physiology , Humans , Leigh Disease/genetics , Molecular Sequence Data , RNA, Messenger/metabolism , Sequence Homology, Nucleic AcidABSTRACT
Cytochrome c oxidase was isolated from turkey liver, heart and breast skeletal muscle and separated by SDS/PAGE. The N-terminal amino-acid sequence of subunit VIa from all tissues and internal sequences from the skeletal muscle enzyme show homology to the mammalian liver-type subunit VIaL, which was verified by isolation and sequencing of the cDNA of turkey subunit VIa. No cDNA corresponding to subunit VIaH (mammalian heart-type) could be found by RACE-PCR with mRNA from all turkey tissues. Measurement of proton translocation with the reconstituted enzymes from turkey liver and heart revealed H+/e- ratios below 0.5 that were independent of the intraliposomal ATP/ADP ratio, as previously found with the bovine liver enzyme. Under identical conditions, the bovine heart enzyme revealed H+/e- ratios of 0.85 at low and 0.48 at high intraliposomal ATP/ADP ratios. The results suggest that in birds the lower H+/e-ratio of cytochrome c oxidase participates in elevated resting metabolic rate and thermogenesis.
Subject(s)
Electron Transport Complex IV/metabolism , Energy Metabolism , Liver/enzymology , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , DNA, Complementary , Electron Transport Complex IV/chemistry , Molecular Sequence Data , Muscle, Skeletal/enzymology , Myocardium/enzymology , Sequence Homology, Amino Acid , TurkeysABSTRACT
The Mohr-Tranebjaerg syndrome (MTS), a neurodegenerative syndrome characterized by progressive sensorineural hearing loss, dystonia, mental retardation and blindness, is a mitochondrial disease caused by mutations in the deafness/dystonia peptide 1 (DDP1) gene. DDP1 shows similarity to the yeast proteins Tim9, Tim10 and Tim12, components of the mitochondrial import machinery for carrier proteins. Here, we show that DDP1 belongs to a large family of evolutionarily conserved proteins. We report the identification, chromosomal localization and expressional analysis of six human family members which represent further candidate genes for neurodegenerative diseases.
