EACH OF 3 POSITIVELY-CHARGED AMINO-ACIDS IN THE C-TERMINAL REGION OF YEAST MITOCHONDRIAL ATP SYNTHASE SUBUNIT-8 IS REQUIRED FOR ASSEMBLY

Each of three conserved positively-charged residues in the C-terminal region of subunit 8 of yeast (Saccharomyces cerevisiae) mitochondrial ATP synthase was replaced with isoleucine. The assembly and functional properties of the resulting variants (substituted at Arg-37, Arg-42 a nd Lys-47) were examined using in-vitro systems to assay import into i solated mitochondria and to monitor assembly into ATP synthase, as wel l as an in-vivo rescue system using host yeast cells lacking endogenou s subunit 8. Each such variant was found to be impaired in assembly in vitro, after import in the form of a chimaeric protein bearing a lead er sequence with mitochondrial targeting function. Import precursors b earing a duplicated-leader sequence, engendering enhanced delivery to mitochondria of the passenger variant subunit-8 proteins, enabled asse mbly of the (Lys-47 --> Ile) variant to be detected in vitro but not t hat of (Arg-37 --> Ile) or (Arg-42 --> Ile) variants. The respiratory growth of subunit 8-deficient host cells could be rescued with the (Ly s-47 --> Ile) variant expressed allotopically in the nucleus. Such res cued cells were found to have an enhanced growth rate (comparable to t hat produced by non-mutagenized parental subunit 8) when delivered to mitochondria with the duplicated-leader sequence, as compared to the s ingle-leader sequence. This confirms that the impediment in the (Lys-4 7 --> Ile) variant lies in the efficiency of its assembly, rather than a functional defect, as such, arising from the loss of that positive charge. In contrast, host cells were unable to be rescued by the (Arg- 37 --> Ile) and (Arg-42 --> Ile) variants, even when they were endowed with the duplicated leader sequence. It is concluded that the positiv ely-charged C-terminal domain of subunit 8, common to fungal and mamma lian homologues of this protein, plays a key role in its assembly into mitochondrial ATP synthase.