Characterization of the alpha and beta-subunits 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-beta-D-maltoside followed by Sepharose Hexyl ammonium chromatography, a matrix that interacts with the F1 sector of mitochondrial ATPases. The alpha-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 alpha-subunit was susceptible to limited proteolytic digestion induced by heat. An endogenous protease removed the first 22 residues of the mature alpha-subunit. Subunit beta was also identified by N-terminal sequencing of 31 residues. This subunit of 63 kDa exhibited a higher apparent molecular mass than alpha, as judged by its mobility on denaturing polyacrylamide gel electrophoresis. This beta-subunit is 7-8 kDa larger than the beta-subunits of other mitochondrial ATPases. It is suggested that the beta-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 beta-subunit of C. reinhardtii showed homology with the endogenous ATPase inhibitors from various 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 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 beta-subunits of the ATP synthases from these algae, may play a regulatory role in these enzymes.
Atteia
A
A
Departamento de Bioenergética, Universidad Nacional Autónoma de México, México, D.F., Mexico.
Dreyfus
G
G
González-Halphen
D
D
D000595:Amino Acid Sequence; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000737:chemistry / Q000201:enzymology; D000460:Chlorophyta / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D009840:Oligomycins / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D014475:Uncoupling Agents / Q000494:pharmacology
D009840:Oligomycins; D010446:Peptide Fragments; D014475:Uncoupling Agents; D010450:Endopeptidases; D006180:Proton-Translocating ATPases