The NDUFA1 gene product (MWFE protein) is essential for activity of complex I in mammalian mitochondria

Abstract

The MWFE polypeptide of mammalian complex I (the proton-translocating NADH-quinone oxidoreductase) is 70 amino acids long, and it is predicted to be a membrane protein. The NDUFA1 gene encoding the MWFE polypeptide is located on the X chromosome. This polypeptide is 1 of approximately 28 "accessory proteins" identified in complex I, which is composed of 42 unlike subunits. It was considered accessory, because it is not one of the 14 polypeptides making up the core complex I; a homologous set of 14 polypeptides can make a fully functional proton-translocating NADH-quinone oxidoreductase in prokaryotes. One MWFE mutant has been identified and isolated from a collection of respiration-deficient Chinese hamster cell mutants. The CCL16-B2 mutant has suffered a deletion that would produce a truncated and abnormal MWFE protein. In these mutant cells, complex I activity is reduced severely (<10%). Complementation with hamster NDUFA1 cDNA restored the rotenone-sensitive complex I activity of these mutant cells to approximately 100% of the parent cell activity. Thus, it is established that the MWFE polypeptide is absolutely essential for an active complex I in mammals.

Figure 1

Alignment of the wild-type and mutant CCL16-B2 cDNA sequences. The wild-type hamster sequence has been submitted to GenBank (accession no. AF100706). The deletion in the mutant is indicated by the dashed line. Translation in the mutant yields a truncated peptide, starting with a wild-type sequence (first 13 amino acids), followed by 16 amino acids from a different reading frame.

Figure 2

Alignment of the available mammalian MWFE amino acid sequences emphasizing the very significant conservation (identity indicated in black) and a striking set of changes around positions 39–44. This polypeptide is not processed proteolytically on import into mitochondria (14).

Figure 3

Northern blot of total RNA from wild-type cells, CCL16-B1, and the two mutant cell lines described here, CCL16-B2 and V79-G4. Equal loading of the gels was verified by both staining and labeling of ribosomal RNAs. From molecular mass markers run in an adjoining lane, the size of the mRNA was estimated to be ≈400 nt. The CCL16-B2 mRNA is slightly smaller and reduced in amount (see Results).

Figure 4

Comparison of rotenone-sensitive NADH-quinone oxidoreductase activities in wild-type (W), mutant CCL16-B2 (M), and a complemented mutant clone transfected with the NDUFA1-cDNA expression vector (C). Determinations were made by measuring oxygen consumption (respiration) that was stimulated by malate plus glutamate and inhibited by rotenone in cells that had been made permeable with digitonin (see Materials and Methods). Bars represent the average of five such measurements, and the standard deviation is indicated.

Figure 5

Immunoblotting analysis of several peripheral membrane proteins of complex I in wild-type (W), mutant CCL16-B2 (M), and transfected (complemented) cells (C). A preparation of bovine heart mitochondrial membranes (B) also is included for comparison. Mitochondrial membranes were prepared as described in Material and Methods. Gels were loaded to obtain comparable signals for the β-subunit of the F₁-ATPase.