Complex IV heterodimeric Cox2 in Polytomella sp: Import and protein-protein interactions

Gene transfer from the mitochondrion to the nucleus is an ongoing evolutionary process, that explains why extant mitochondrial genomes are highly reduced, encoding only a few set of proteins required for oxidative phosphorylation. These proteins exhibit high hydrophobicity, so it is believed that the functional migration of the corresponding genes to the nucleus has been limited by this parameter. However, the chlorophycean algal lineage, that includes the colorless alga Polytomella sp., has a mitochondrial DNA lacking the cox2 gene that encodes subunit II (COX2) of cytochrome c oxidase. In Polytomella sp. this gene was split into two genes, cox2a and cox2b, which encode proteins COX2A and COX2B respectively. A feature of these subunits is an additional sequences located at one of its ends, at the N-terminal region for COX2B and at the C-terminal region for COX2A. These extensions do not have similarity to any other COX2 sequence reported so far. In addition, COX2A has a pre-sequence of 130 amino acids in theN-terminal region. It has been proposed that the extensions are involved in the formation of the heterodimer COX2A/COX2B and in its structural stabilization. In order to characterize the interaction of proteins COX2A and COX2B and to determine the importance of the extensions, interaction assays were performed, such as affinity chromatography and Far-Western blotting with the recombinant proteins. The results indicate that the COX2B extension is necessary but not essential for the association of COX2A/COX2B. Both proteins are synthesized in the cytosol, imported and assembled, so we explored this process in vitro, using isolated Polytomella sp. mitochondria. Our results suggest that COX2B is imported directly into the intermembrane space, while COX2A follows an energy-dependent import pathway. In addition, the MTS of the COX2A precursor is edited. This is the first time that the in vitro import of split COX2 subunits into mitochondria has been achieved. Work supported by CONACyT (128110) and DGAPA-UNAM (IN 203311).