Bioenergetic Analysis of FLAG Tagged-Subunit 8 of Saccharomyces cerevisiae Mitochondrial ATP Synthase

Abstract

The majority of cellular energy in the form of adenosine triphosphate (ATP) is synthesized by the F F -ATP synthase. The yeast mitochondrial F F -ATPsynthase is a multisubunit complex that contains at least 17 different subunits grouped into F and F sectors. Subunit 8 of yeast mitochondrialATP synthase is a hydrophobic protein of 48 amino acids encoded by the mitochondrial gene. Subunit 8 has three distinct domains; an N-terminal domain, a central hydrophobic domain and a C-terminal domain. FLAG tag addition to the C-terminus of subunit 8 and its variants has facilitated elucidation of subunit 8's membrane topology. In order to analyze its detailed structure and function, a set of strains expressing FLAG tagged-subunit 8 and its variants were subjected to bioenergetic analysis at cellular and mitochondrial levels. Results obtained showed that the hydrophobic character of the central hydrophobic domain of subunit 8 is essential for functional coupling between F and F sectors, hence for mitochondrialATPsynthase function.