Defining redox centers in human electron transfer flavoprotein: ubiquinone oxidoreductase (ETF:QO) by expression in Saccharomyces cerevisiae
- Univ. of Colorado School of Medicine, Denver, CO (United States)
Mutations in ETF or ETC:QO cause glutaric acidemia type II (GA2). ETF:QO is an iron-sulfur flavoprotein in the inner mitochondrial membrane which transfers electrons from ETF in the mitochondrial matrix to ubiquinone (Q). The human ETF:QO gene is on chromosome 4q32{r_arrow}qter, and encodes a 617 amino acid precursor which is processed to the 64 kDa mature form in the mitochondrion. One ETF:QO mutation in GA2 is a G{r_arrow}T transversion in a donor splice site, deleting the 222 bp upstream exon from the transcript. The deleted 74 amino acids are near the carboxyl terminus just beyond a predicted membrane helix, and include C561, one of four cysteine residues predicted to ligate the 4Fe4S cluster. The mutant protein is not stable in patient fibroblasts. We have expressed cDNAs encoding wild type (wt) ETF:QO, ETF:QO with the 74 amino acid deletion, and ETFF:QO with only a C561A mutation, in S cerevisiae. In all instances, precursor and mature ETF:QOs were stably inserted into the mitochondrial membrane. ETF:QO (C561A) is extracted from the membrane under the same conditions as wt ETF:QO, but ETF:QO with the deletion is much more difficult to extract. Wt ETF:QO accepts electrons from ETF and reduces Q but, while both mutant proteins accept electrons from ETF, neither of them reduces Q. This work demonstrates that C561 in human ETF:QO is essential for Q reduction (probably because it ligands the 4Fe4S cluster), that mutant proteins that are unstable in man may be stable in other systems, that cleavage of signal peptide from precursor proteins can occur within the inner mitochondrial membrane, and the general usefulness of expressing human mitochondrial proteins in yeast.
- OSTI ID:
- 133968
- Report Number(s):
- CONF-941009--
- Journal Information:
- American Journal of Human Genetics, Journal Name: American Journal of Human Genetics Journal Issue: Suppl.3 Vol. 55; ISSN AJHGAG; ISSN 0002-9297
- Country of Publication:
- United States
- Language:
- English
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