Abstract :
[en] The F O F1-ATP synthase (EC3.6.3.14) produces most of the cellular ATP in aerobic conditions. This enzyme complex is found in energy transducing membranes such as the mitochondrial inner membrane, the thylakoid membrane of the chloroplast, and the bacterial plasma membrane. The Escherichia coli ATP synthase exhibits the simplest known subunit composition, which consists of eight subunits α3 β3 γ1 δ1 ε1 a 1 b 2 c 10-12. Subunits -α, -β, -γ, -δand -εform the F 1 domain, while subunits -a, -b and -c form the F O domain. The mitochondrial F O F 1 -ATP synthase is more complex than the bacterial enzyme and is composed of at least 15 different subunits. Besides having orthologs of the eight essential subunits found in E. coli, it contains several supernumerary subunits (<20 KDa), which are required for the stability or the regulation of the enzymatic complex, while others are involved in its dimerization or oligomerization. In this regard, and in contrast to other known enzyme complexes, the ATPase from the algae Chlamydomonas reinhardtii and Polytomella sp. show unique structural characteristics. The algal enzyme was found to have orthodox subunits of the catalytic domain (-αand-β), as well as those that are part of the central rotary stalk (-γ, -δ, -ε and -c), however, with respect to the subunits which constitute the peripheral arm, only subunits –a and –OSCP were found. In addition, nine subunits of unknown evolutionary origin that do not have clear homologs in the databases were also found. These subunits were named ASA (Mitochondrial ATP Synthase Associated Protein) and were numbered successively as ASA1-9 subunits. It is thought that the ASA subunits replace those involved in the formation of the peripheral arm (-b, -d, -e, -f, -g, -IF1, -A6L and -F6), the dimerization of the complex (-e, -g) and the regulation (-IF1) of the enzyme activity. Therefore, these ASA subunits could be the main components of the peripheral arm (ASA1, ASA2, ASA3, ASA4, and ASA7), others may participate in the dimerization of the complex (ASA6 and ASA9) and others could play a regulatory role (ASA?).Recent work in the laboratory using methodologies such as dissociation of the enzyme into sub-complexes induced by high temperatures, treatment with crosslinking agents or association studies employing recombinant proteins, have addressed the study of the interactions of the ASA subunits in the ATP synthase of Polytomella sp. However, still little is known about subunits -a, -c, ASA5, ASA6, ASA8 and ASA9 that form the membrane fraction of the complex. These subunits probably have a transmembrane domain or highly hydrophobic regions. Therefore, it is our interest to approach the study of the interactions of the hydrophobic subunits of the peripheral arm of the ATP synthase and to obtain more information about the membrane domain of this enzymatic complex. In this work we present the results obtained using recombinant proteins and their purification for the subsequent formation of in vitro sub-complexes, as well as qualitative information obtained from interaction assays using the two hybrid system. For the use of recombinant proteins it is necessary to clone the genes that encode for the mature subunits:ASA6, ASA8 and ASA9 from the alga ATP synthase into vectors for further overexpression in E. coli and purifying them by fast protein liquid chromatography (FPLC). The recombinant proteins were used for interaction assays such as type Far-Western blotting and co-purification by immobilized metal affinity chromatography (IMAC) using nickel-NTA (nitrilotriacetic acid).The in vivo interaction assays were performed using yeast two-hybrid system. For this purpose, the constructions of the genes encoding the ASA6, ASA8 and ASA9 subunits vectors corresponding to the activation domain (pAD) and binding domain (pBD) were obtained. Subsequently, interaction assays were performed in the medium called "low stringency" (-Leu/-Trp/-His) and "high stringency" (-Leu/-Trp/-His/- Ade) observing interactions only in the former conditions. The results suggest an ASA6-ASA6 interaction, and in less degree the interactions ASA9-ASA8 and ASA9-ASA9. We will confirm these data using other methodologies used to determine protein-protein interactions. Work supported by CONACyT (128110) and DGAPA-UNAM (IN 203311).
