Each of three 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 and Lys-47) were examined using in-vitro systems to assay import into isolated mitochondria and to monitor assembly into ATP synthase, as well as an in-vivo rescue system using host yeast cells lacking endogenous 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 leader sequence with mitochondrial targeting function. Import precursors bearing a duplicated-leader sequence, engendering enhanced delivery to mitochondria of the passenger variant subunit-8 proteins, enabled assembly of the (Lys-47-->Ile) variant to be detected in vitro but not that of (Arg-37-->Ile) or (Arg-42-->Ile) variants. The respiratory growth of subunit 8-deficient host cells could be rescued with the (Lys-47-->Ile) variant expressed allotopically in the nucleus. Such rescued cells were found to have an enhanced growth rate (comparable to that produced by non-mutagenized parental subunit 8) when delivered to mitochondria with the duplicated-leader sequence, as compared to the single-leader sequence. This confirms that the impediment in the (Lys-47-->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 positively-charged C-terminal domain of subunit 8, common to fungal and mammalian homologues of this protein, plays a key role in its assembly into mitochondrial ATP synthase.
Papakonstantinou
T
T
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Galanis
M
M
Nagley
P
P
Devenish
R J
RJ
D001483:Base Sequence; D001692:Biological Transport / Q000502:physiology; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021382:Protein Sorting Signals / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D006180:Proton-Translocating ATPases