Isolation and characterization of the mitochondrial ATP synthase fromChlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the ? subunit

Abstract

We have isolated the F0F1-ATP synthase complex from oligomycin-sensitive mitochondria of the green alga Chlamydomonas reinhardtii. A pure and active ATP synthase was obtained by means of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and PMSF [corrected]. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase alpha subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58-70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase beta subunit from C. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771-780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15-18 residues longer than in ATP synthase alpha subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.

The Typically Mitochondrial DNA-encoded ATP6 Subunit of the F1F0-ATPase Is Encoded by a Nuclear Gene in Chlamydomonas reinhardtii

“…3). The three major subunits were identified as subunits ␣, ␤, and ␥, since their apparent molecular masses are similar to those reported previously (31). Their identities were confirmed by N-terminal sequence analysis of their first five amino acids.…”

New Insights into the Unique Structure of the F0F1-ATP Synthase from the Chlamydomonad Algae Polytomella sp. and Chlamydomonas reinhardtii

“…and its photosynthetic counterpart C. reinhardtii share an atypical polypeptide composition. The unusual extensions that C. reinhardtii exhibit at the N and C termini of the catalytic subunits-a and -b, respectively (Franzén and Falk, 1992;Nurani and Franzén, 1996), are shown to be also present in Polytomella sp. The fact that the a-and b-subunit extensions of both algae are highly conserved suggests functional restraints.…”

“…First, the catalytic subunits-a and -b in the green alga Chlamydomonas reinhardtii exhibit peculiar extensions at their N and C termini, respectively (Franzén and Falk, 1992;Nurani and Franzén, 1996). The a-subunit in the nonphotosynthetic alga Polytomella sp.…”

The Mitochondrial Oxidative Phosphorylation Proteome of Chlamydomonas reinhardtii Deduced from the Genome Sequencing Project

“…doe.gov; Shrager et al, 2003). Moreover, the recent development of procedures to isolate Chlamydomonas mitochondria almost free of thylakoid contaminants (Eriksson et al, 1995;Nurani and Franzen, 1996;Cardol et al, 2002), to separate the major mitochondrial complexes in BN-PAGE (van Lis et al, 2003;Cardol et al, 2004), and to analyze by bioinformatics the algalnucleic acid sequences (http://www.chlamy.org/) allowed us to initiate the characterization of the algal OXPHOSome Cardol et al, 2004).…”