Bioenergetic and structural consequences of allotopic expression of subunit 8 of yeast mitochondrial ATP synthase - The hydrophobic character of residues 23 and 24 is essential for maximal activity and structural stability of the enzyme complex

Subunit 8 (Y8), a mitochondrially encoded subunit of the F-0 sector of the F1F0-ATP synthase is essential for oxidative phosphorylation. We have previ ously introduced the technique of allotopic expression to study the structu re/function of Y8, whereby an artificial Y8 gene is expressed in the nucleu s of cells lacking a functional mitochondrial Y8, thus generating assembly of a functional F1F0-ATPase complex. In this paper we show that when a gene encoding an essentially unmodified version of Y8 is allotopically expresse d, ATP synthesis and hydrolysis rates, as well as efficiency of oxidative p hosphorylation, were similar to those of the parental wild-type strain in w hich Y8 is naturally expressed in mitochondria. We then tested the requirem ent for the hydrophobicity of the central domain (residues 14-32), which po ssibly represents a transmembrane stem, by introducing adjacent negative ch arges at different positions of Y8. One of the variants thus generated, whi ch carries the double substitution Leu23-->Asp, Leu24-->Asp, when expressed in a strain lacking endogenous Y8, gave rise to cells which grew very slow ly by oxidative phosphorylation. Measurement of bioenergetic parameters sho wed two major defects in these cells relative to control cells allotopicall y expressing unmodified Y8. First, the activity of the F1F0-ATP synthase wa s significantly decreased. ATP synthesis and state 3 of respiration were re duced by approximate to 30-40%. ATP hydrolysis was reduced by approximate t o 30% and was almost insensitive to the F-0 inhibitor oligomycin. Second, t he physical coupling between the two sectors of the enzyme, as well as the stability of the F-1 sector itself, were affected as shown by decreased rec overy of F-0 sector [8, 9, b, oligomycin sensitivity-conferring protein (OS CP), d, h and f] and F-1 sector (alpha, gamma, delta) subunits in immunopre cipitates of ATP synthase. This study indicates that Y8 not only performs a n important role in the structure of the mitochondrial complex but also in its activity. We conclude that the hydrophobic character of amino acids 23 and 24 in the middle of the putative transmembrane stern of Y8 is essential for coupling proton transport through F-0 to ATP synthesis on F-1.