Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico.
Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210.
Mol Biol Cell. 2019 Aug 15;30(18):2358-2366. doi: 10.1091/mbc.E18-12-0788. Epub 2019 Jul 18.
Experimentally relocating mitochondrial genes to the nucleus for functional expression (allotopic expression) is a challenging process. The high hydrophobicity of mitochondria-encoded proteins seems to be one of the main factors preventing this allotopic expression. We focused on subunit II of cytochrome oxidase (Cox2) to study which modifications may enable or improve its allotopic expression in yeast. Cox2 can be imported from the cytosol into mitochondria in the presence of the W56R substitution, which decreases the protein hydrophobicity and allows partial respiratory rescue of a -null strain. We show that the inclusion of a positive charge is more favorable than substitutions that only decrease the hydrophobicity. We also searched for other determinants enabling allotopic expression in yeast by examining the gene in organisms where it was transferred to the nucleus during evolution. We found that naturally occurring variations at within-membrane residues in the legume Cox2 could enable yeast allotopic expression. We also evidence that directing high doses of allotopically synthesized Cox2 to mitochondria seems to be counterproductive because the subunit aggregates at the mitochondrial surface. Our findings are relevant to the design of allotopic expression strategies and contribute to the understanding of gene retention in organellar genomes.
将线粒体基因实验性转移到细胞核中进行功能表达（异位表达）是一个具有挑战性的过程。线粒体编码蛋白的高度疏水性似乎是阻止这种异位表达的主要因素之一。我们专注于细胞色素氧化酶亚基 II（Cox2）的研究，以探讨哪些修饰可以使其在酵母中实现或改善异位表达。在 W56R 取代的情况下，Cox2 可以从细胞质中导入线粒体，该取代降低了蛋白质的疏水性，并允许-缺失菌株的部分呼吸恢复。我们表明，带正电荷的插入比仅降低疏水性的取代更有利。我们还通过检查在进化过程中已将其转移到核中的生物体中的 基因，来寻找其他可在酵母中实现异位表达的决定因素。我们发现豆科植物 Cox2 中膜内残基的自然变异可以使酵母实现异位表达。我们还证明，将异位合成的 Cox2 高剂量导向线粒体似乎适得其反，因为亚基在线粒体表面聚集。我们的发现与异位表达策略的设计有关，并有助于理解细胞器基因组中基因的保留。
