Characterization of the α and β-subunits of the F 0F 1-ATPase from the alga Polytomella spp., a colorless relative of Chlamydomonas reinhardtii

Abstract

The isolation and partial characterization of the oligomycin-sensitive F 0F 1-ATP synthase/ATPase from the colorless alga Polytomella spp. is described. Purification was performed by solubilization with dodecyl- β- d-maltoside followed by Sepharose Hexyl ammonium chromatography, a matrix that interacts with the F 1 sector of mitochondrial ATPases. The α-subunit, which migrates on SDS-polyacrylamide gels with an apparent molecular mass of 55 kDa, was identified by the N-terminal sequencing of 47 residues. This subunit exhibited a short extension at its N-terminus highly similar to the one described for the unicellular alga Chlamydomonas reinhardtii (Nurani, G. and Franzén L.-G. (1996) Plant Mol. Biol. 31, 1105–1116). In whole mitochondria, the α-subunit was susceptible to limited proteolytic digestion induced by heat. An endogenous protease removed the first 22 residues of the mature α-subunit. Subunit β was also identified by N-terminal sequencing of 31 residues. This subunit of 63 kDa exhibited a higher apparent molecular mass than α, as judged by its mobility on denaturing polyacrylamide gel electrophoresis. This β-subunit is 7–8 kDa larger than the β-subunits of other mitochondrial ATPases. It is suggested that the β-subunit from Polytomella spp. may have a C-terminal extension similar to that described for the green alga C. reinhardtii (Franzén, L.-G. and Falk, G. (1992) Plant Mol. Biol. 19, 771–780). In addition, it was found that the C-terminal extension of the β-subunit of C. reinhardtii showed homology with the endogenous ATPase inhibitors from various sources and with the ϵ-subunit from the F 0F 1-ATP synthase from Escherichia coli, which is considered to be a functional homolog of the inhibitor proteins. The data reported here provide the first biochemical evidence for a close relationship between the colorless alga Polytomella spp. and its photosynthetic counterpart C. reinhardtii. It is also suggested that the C-terminal extensions of the β-subunits of the ATP synthases from these algae, may play a regulatory role in these enzymes.