Site directed mutagenesis of subunit 8 of yeast mitochondrial ATP synthase

Abstract

The function of the positively charged C‐terminal region of mitochondrially encoded subunit 8 of yeast mitochondrial ATP synthase was investigated using derivatives truncated at each of the 3 positively charged residues (Arg37, Arg42 and Lys47). Each construct, allotopically expressed in the nucleus, was tested for its ability to import and assemble functionally into ATP synthase in yeast cells unable to synthesize mitochondrial subunit 8. The efficiency of import of each construct into isolated wild‐type yeast mitochondria was also determined. One construct truncated at the penultimate residue of subunit 8 (Lys47) functions in vivo and shows efficient import in vitro. Thus subunit 8 can function with only two positively charged residues. The remainder of the subunit 8 variants failed to rescue in vivo. Since they all show greatly reduced or undetectable import in vitro, presumably because of the increased hydrophobic character of the subunit 8 moiety in the chimaeric precursors, the status of these variants as regards assembly and function is not clear.

Structure/Function Analysis of Yeast Mitochondrial ATP Synthase Subunit 8a

“…Further truncation of Y8 at the positions of the other two positive charges gave rise to the precursors N9L/Y8-1(Arg42 + STP) and N9L/Y8-1(Arg37 + STP), which both failed to rescue subunit 8-deficient cells in vivaz3 Significantly, neither precursor could be imported in vitro. 23 These variants, unfortunately, did not provide a useful means for the study of the assembly or function of Y8.…”

“…The results eliminate the possibility that removal of the two most distal residues, as such, from full-length Y8 contribute to the altered assembly properties Considering the variants N9L/Y8-1(Arg42 + Ile) and N9L/Y8-1(Arg37 .+ Ile), both are shown to be able to be imported into isolated mitochondria (TABLE I), thus overcoming the gross import deficiencies of the corresponding truncation variants. 23 Interestingly, evidence of degradation of the both processed Y8-1 variants during the import reaction suggests that they are remaining accessible to intramitochondrial proteases; in contrast, the Y8-1 parent and Yg-l(Lys47 + Ile) are apparently delivered to a location that is protease inaccessible. The Y&l(Arg42 + Ile) and Yg-l(Arg37 + Ile) variants are severely affected in terms of their assembly in vitro (TABLE 1).…”