A high copy suppressor screen identifies factors enhancing the allotopic production of subunit II of cytochrome c oxidase.

Abstract

Allotopic expression refers to the artificial relocation of an organellar gene to the nucleus. Subunit 2 (Cox2) of cytochrome c oxidase, a subunit with two transmembrane domains (TMS1 and TMS2) residing in the inner mitochondrial membrane with a Nout-Cout topology, is typically encoded in the mitochondrial cox2 gene. In the yeast Saccharomyces cerevisiae, the cox2 gene can be allotopically expressed in the nucleus, yielding a functional protein that restores respiratory growth to a Δcox2 null mutant. In addition to a mitochondrial targeting sequence followed by its natural 15-residue leader peptide, the cytosol synthesized Cox2 precursor must carry one or several amino acid substitutions that decrease the mean hydrophobicity of TMS1 and facilitate its import into the matrix by the TIM23 translocase. Here, using a yeast strain that contains a COX2W56R gene construct inserted in a nuclear chromosome, we searched for genes whose overexpression could facilitate import into mitochondria of the Cox2W56R precursor and increase respiratory growth of the corresponding mutant strain. A COX2W56R expressing strain was transformed with a multicopy plasmid genomic library, and transformants exhibiting enhanced respiratory growth on non-fermentable carbon sources were selected. We identified three genes whose overexpression facilitates the internalization of the Cox2W56R subunit into mitochondria, namely: TYE7, RAS2 and COX12. TYE7 encodes a transcriptional factor, RAS2 a GTP-binding protein, and COX12 a non-core subunit of cytochrome c oxidase. We discuss potential mechanisms by which the TYE7, RAS2 and COX12 gene products could facilitate the import and assembly of the Cox2W56R subunit produced allotopically.