Membrane Topology of Subunit

The yeast mitochondrial F1F0-ATP synthase is a multisubunit complex that contains at least 17 different subunits. Subunit 8 of yeast mitochondrial ATP synthase is a hydrophobic protein of 48 amino acids encoded by the mitochondrial ATP8 gene. There is no homologue of subunit 8 found in bacteria. Subunit 8 has three distinct domains; an N-terminal domain, a central hydrophobic domain and a C-terminal domain. Subunit 8 has been shown to adopt a transmembrane topology with the central hydrophobic domain spans the inner mitochondrial membrane once. In order to elucidate the need of subunit 8 to maintain transmembrane topology for its functioning, a severely functionally defective subunit 8 variant that has been introduced with double-charged residues within the central hydrophobic domain was analysed. A gene encoding this variant was expressed in a yeast strain lacking endogenous subunit 8. The subunit 8 variant was then targeted into mitochondria. Following its assembly into mitochondrial ATP synthase complex, its membrane topology was determined. The results obtained showed that subunit 8 was obligatory to maintain a transmembrane topology for providing proper functioning. The transmembrane topology may be critical for subunit 8’s proposed structural roles as part of the stator stalk of the mitochondrial ATP synthase complex. Key words: membrane topology, ATP synthase, mitochondria, yeast ATP synthase, also known as F0F1-ATPase, is a large complex of 600 kDa that uses the proton electrochemical gradient generated by the respiratory chain to catalyse ATP synthesis from ADP and Pi (Thomas et al. 2008). The yeast mitochondrial ATP synthase is a multisubunit complex composed of at least 17 subunits grouped into two sectors, a membrane-extrinsic sector (F1) and a membrane-embedded sector (F0). The two sectors are linked by protein stalks. The F1 sector is comprised of five subunits a3, b3, g1, d1 and e1, and is coupled to proton flux through the F0 sector. The F0 sector spans the membrane and is composed of subunits b