Dissecting the peripheral arm of the mitocondrial ATP synthase of chlorophycean algae.

Mitochondrial F 1 Fo-ATP synthase (complex V) makes ATP using the electrochemical proton gradient generated by the respiratory chain. It is an oligomeric complex embedded in the inner mitochondrial membrane that works like a rotary motor. Chlamydomonas reinhardtii and Polytomella sp., two members of the chlorophycean lineage of unicellular green algae, have a highly-stable dimeric mitochondrial F 1 Fo-ATP synthase, with an estimated molecular mass of 1600 kDa. The chlorophycean enzyme contains the eight conserved polypeptides present in the vast majority of eukaryotes that represent the main components of the proton-driven rotary motor and the catalytic sector of the enzyme: subunits alpha, beta, gamma, delta, epsilon, a (ATP6), c (ATP9), and OSCP. Nevertheless, and in sharp contrast with other mitochondrial F 1 Fo-ATP synthases like the one from beef heart, the algal enzyme seems to lack several classic components: the subunits of the peripheral stalk b, d, f, A6L, and F6, the subunits responsible for dimer formation e and g, and the regulatory polypeptide IF 1 . Instead, the algal enzyme contains nine subunits with molecular masses ranging from 8 to 60 kDa named ASA1 to ASA9 (for ATP Synthase Associated proteins). These polypeptides have no clear orthologs in the databases and seem to be unique to chlorophycean algae. The nine ASA subunits build up a highly-robust peripheral stalk with a unique architecture, as observed on single-particle electron microscopy (EM) images.
Our group has found of interest to gain further insights on the close-neighbor relationships between the ASA subunits and their interactions with some of the classical subunits. We have therefore explored with some detail the topological disposition of the components of the algal mitochondrial ATP synthase using different experimental approaches: detection of subunit-subunit interactions based on cross-linking experiments, the yeast two hybrid system or reconstitution with recombinant subunits; generation of sub-complexes after partial dissociation of the dimeric ATP synthase; inference of subunit stoichiometry based on labelling of cysteine residues and modelling of the overall structural features of the complex from small-angle X ray scattering data and EM image reconstruction. Based on the results obtained from these diverse experimental strategies, we suggest a refined model for the topological disposition of the 17 polypeptides that constitute the algal mitochondrial ATP synthase.