CHARACTERIZATION OF THE ALPHA-SUBUNIT AND BETA-SUBUNIT OF THE F0F1-ATPASE FROM THE ALGA POLYTOMELLA SPP, A COLORLESS RELATIVE OF CHLAMYDOMONAS-REINHARDTII

The isolation and partial characterization of the oligomycin-sensitive F0F1-ATP synthase/ATPase from the colorless alga Polytomella spp. is described. Purification was performed by solubilization with dodecyl-b eta-D-maltoside followed by Sepharose Hexyl ammonium chromatography, a matrix that interacts with the F-1 sector of mitochondrial ATPases. T he alpha-subunit, which migrates on SDS-polyacrylamide gels with an ap parent molecular mass of 55 kDa, was identified by the N-terminal sequ encing of 47 residues. This subunit exhibited a short extension at its N-terminus highly similar to the one described for the unicellular al ga Chlamydomonas reinhardtii (Nurani, G. and Franzen L.-G. (1996) Plan t Mel. Biol. 31, 1105-1116). In whole mitochondria, the alpha-subunit was susceptible to limited proteolytic digestion induced by heat. An e ndogenous protease removed the first 22 residues of the mature alpha-s ubunit. Subunit beta was also identified by N-terminal sequencing of 3 1 residues. This subunit of 63 kDa exhibited a higher apparent molecul ar mass than alpha, as judged by its mobility on denaturing polyacryla mide gel electrophoresis. This beta-subunit is 7-8 kDa larger than the beta-subunits of other mitochondrial ATPases. It is suggested that th e beta-subunit from Polytomella spp. may have a C-terminal extension s imilar to that described for the green alga C. reinhardtii (Franzen, L .-G. and Falk, G. (1992) Plant Mol. Biol. 19, 771-780). In addition, i t was found that the C-terminal extension of the beta-subunit of C. re inhardtii showed homology with the endogenous ATPase inhibitors from v arious sources and with the epsilon-subunit from the F0F1-ATP synthase from Escherichia coli, which is considered to be a functional homolog of the inhibitor proteins. The data reported here provide the first b iochemical evidence for a close relationship between the colorless alg a Polytomella spp. and its photosynthetic counterpart C. reinhardtii. It is also suggested that the C-terminal extensions of the beta-subuni ts of the ATP synthases from these algae, may play a regulatory role i n these enzymes.