Nguyen Theresa P T, Casarin Alberto, Desbats Maria Andrea, Doimo Mara, Trevisson Eva, Santos-Ocaña Carlos, Navas Placido, Clarke Catherine F, Salviati Leonardo
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1569, USA; Molecular Biology Institute, University of California, Los Angeles, CA 90095-1569, USA.
Clinical Genetics Unit, Dept. of Pediatrics, University of Padova, Via Giustiniani 3, Padova 35128, Italy.
Biochim Biophys Acta. 2014 Nov;1841(11):1628-38. doi: 10.1016/j.bbalip.2014.08.007. Epub 2014 Aug 23.
Coq5 catalyzes the only C-methylation involved in the biosynthesis of coenzyme Q (Q or ubiquinone) in humans and yeast Saccharomyces cerevisiae. As one of eleven polypeptides required for Q production in yeast, Coq5 has also been shown to assemble with the multi-subunit complex termed the CoQ-synthome. In humans, mutations in several COQ genes cause primary Q deficiency, and a decrease in Q biosynthesis is associated with mitochondrial, cardiovascular, kidney and neurodegenerative diseases. In this study, we characterize the human COQ5 polypeptide and examine its complementation of yeast coq5 point and null mutants. We show that human COQ5 RNA is expressed in all tissues and that the COQ5 polypeptide is associated with the mitochondrial inner membrane on the matrix side. Previous work in yeast has shown that point mutations within or adjacent to conserved COQ5 methyltransferase motifs result in a loss of Coq5 function but not Coq5 steady state levels. Here, we show that stabilization of the CoQ-synthome within coq5 point mutants or by over-expression of COQ8 in coq5 null mutants permits the human COQ5 homolog to partially restore coq5 mutant growth on respiratory media and Q6 content. Immunoblotting against the human COQ5 polypeptide in isolated yeast mitochondria shows that the human Coq5 polypeptide migrates in two-dimensional blue-native/SDS-PAGE at the same high molecular mass as other yeast Coq proteins. The results presented suggest that human and Escherichia coli Coq5 homologs expressed in yeast retain C-methyltransferase activity but are capable of rescuing the coq5 yeast mutants only when the CoQ-synthome is assembled.
Coq5催化人类和酿酒酵母中辅酶Q（Q或泛醌）生物合成过程中唯一的C-甲基化反应。作为酵母中Q合成所需的11种多肽之一，Coq5也已被证明能与称为CoQ合成体的多亚基复合物组装在一起。在人类中，几个COQ基因的突变会导致原发性Q缺乏，而Q生物合成的减少与线粒体、心血管、肾脏和神经退行性疾病有关。在本研究中，我们对人类COQ5多肽进行了表征，并检测了其对酵母coq5点突变体和缺失突变体的互补作用。我们发现人类COQ5 RNA在所有组织中均有表达，且COQ5多肽与线粒体内膜基质侧相关联。先前在酵母中的研究表明，保守的CoQ5甲基转移酶基序内或其附近的点突变会导致Coq5功能丧失，但不会导致Coq5稳态水平的改变。在此，我们表明，在coq5点突变体内稳定CoQ合成体或通过在coq5缺失突变体中过表达COQ8，可使人COQ5同源物部分恢复coq5突变体在呼吸培养基上的生长以及Q6含量。对分离的酵母线粒体中的人类COQ5多肽进行免疫印迹分析表明，人类Coq5多肽在二维蓝色天然/SDS-PAGE中的迁移分子量与其他酵母Coq蛋白相同。本文给出的结果表明，在酵母中表达的人类和大肠杆菌CoQ5同源物保留了C-甲基转移酶活性，但只有在CoQ合成体组装好时才能拯救coq5酵母突变体。
