Details

Autor(en) / Beteiligte

• Hotra, Adam
• Suter, Manuel
• Biukovic, Goran
• Ragunathan, Priya
• Kundu, Subhashri
• Dick, Thomas
• Grueber, Gerhard

Titel

Deletion of a unique loop in the mycobacterial F-ATP synthase gamma subunit sheds light on its inhibitory role in ATP hydrolysis-driven H super(+) pumping

Ist Teil von

• The FEBS journal, 2016-05, Vol.283 (10), p.1947-1961

Erscheinungsjahr

2016

Quelle

Wiley-Blackwell Journals

Beschreibungen/Notizen

• The F sub(1)F sub(O)-ATP synthase is one of the enzymes that is essential to meet the energy requirement of both the proliferating aerobic and hypoxic dormant stages of the life cycle of mycobacteria. Most F-ATP synthases consume ATP in the alpha sub(3): beta sub(3) headpiece to drive the gamma subunit, which couples ATP cleavage with proton pumping in the c ring of F sub(O) via the bottom of the gamma subunit. ATPase-driven H super(+) pumping is latent in mycobacteria. The presence of a unique 14 amino acid residue loop of the mycobacterial gamma subunit has been described and aligned in close vicinity to the c-ring loop Priya R et al. (2013) J Bioenerg Biomembr 45, 121-129 Here, we used inverted membrane vesicles (IMVs) of fast-growing Mycobacterium smegmatis and a variety of covalent and non-covalent inhibitors to characterize the ATP hydrolysis activity of the F-ATP synthase inside IMVs. These vesicles formed a platform to investigate the function of the unique mycobaterial gamma loop by deleting the respective loop-encoding sequence ( gamma sub(166-179)) in the genome of M. smegmatis. ATP hydrolysis-driven H super(+) pumping was observed in IMVs containing the Delta gamma sub(166-179) mutant protein but not for IMVs containing the wild-type F-ATP synthase. In addition, when compared to the wild-type enzyme, IMVs containing the Delta gamma sub(166-179) mutant protein showed increased ATP cleavage and lower levels of ATP synthesis, demonstrating that the loop affects ATPase activity, ATPase-driven H super(+) pumping and ATP synthesis. These results further indicate that the loop may affect coupling of ATP hydrolysis and synthesis in a different mode. F-ATP synthases possess in addition to their synthase an ATP-hydrolase activity which is coupled to proton-pumping. The mycobacterial enzyme lacks this reverse activity, enabling the bacterium to survive. Here, studies on the unique subunit gamma -loop of the mycobacterial F-ATP synthase demonstrate that the loop affects ATPase activity, ATPase driven H super(+)-pumping and ATP synthesis.