Novel subfamily of mitochondrial HMG box-containing proteins: functional analysis of Gcf1p from Candida albicans.
Novel subfamily of mitochondrial HMG box-containing proteins: functional analysis of Gcf1p from Candida albicans.
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DOI：
10.1099/mic.0.025759-0
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发表时间：
2009-04
期刊：
影响因子：
1.5
通讯作者：
Katarina Visacka;J. Gerhold;J. Petrovicová;Slavomír Kinský;Priit Jõers;J. Nosek;J. Sedman;L. Tomáška
中科院分区：
文献类型：
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作者：
Katarina Visacka;J. Gerhold;J. Petrovicová;Slavomír Kinský;Priit Jõers;J. Nosek;J. Sedman;L. Tomáška
真核生物的线粒体包含DNA分子群体，这些DNA分子被包装成称为线粒体类核（mt-nucleoids）的高级结构。在酿酒酵母中，线粒体DNA（mtDNA）压缩成线粒体类核主要由含高迁移率族（HMG）盒的蛋白Abf 2介导，其在mtDNA的稳定和代谢中起重要作用。虽然很明显，类似的蛋白质必须存在于其他酵母物种，一个明显的快速分歧率排除了他们的鉴定，表征和比较分析。使用致病性酵母菌白色念珠菌的全基因组序列的计算机分析，我们预测，ORF 19.400/19.8030指定为GCF 1编码一个假定的线粒体HMG盒含蛋白。与包含两个HMG盒的Abf 2 p相比，Gcf 1 p仅包含一个C-末端HMG盒。此外，它包含一个假定的卷曲螺旋结构域，在蛋白质二聚化中具有潜在的作用。荧光显微镜分析的C-末端标记的Gcf 1 p与绿色荧光蛋白（GFP）揭示了其线粒体定位在两个异源（S。cerevisiae）和天然（C.白色念珠菌）宿主。DNA结合特性的生化分析表明，Gcf 1 p是，类似于Abf 2 p，一种非特异性的DNA结合蛋白。为了分析Gcf 1 p在mtDNA代谢中的作用，我们构建了缺失GCF 1基因的一个功能等位基因并在MET 3启动子控制下携带一个GCF 1等位基因的菌株。在抑制条件下，该菌株表现出超过3000倍的GCF 1 mRNA的水平下降，这是与大量减少的mtDNA拷贝数以及重组中间体。Gcf 1 p水平降低对mtDNA代谢的显著影响表明，该蛋白质参与了与线粒体基因组相关的重要分子交易。
Mitochondria of eukaryotic organisms contain populations of DNA molecules that are packed into higher-order structures called mitochondrial nucleoids (mt-nucleoids). In Saccharomyces cerevisiae, the compaction of mitochondrial DNA (mtDNA) into mt-nucleoids is mediated primarily by the high-mobility group (HMG) box-containing protein Abf2, which is an important player in stabilization and metabolism of mtDNA. Although it is evident that analogous proteins must exist in other yeast species, an apparently fast divergence rate has precluded their identification, characterization and comparative analysis. Using in silico analysis of the complete genome sequence of the pathogenic yeast Candida albicans we predicted that the ORF 19.400/19.8030 assigned as GCF1 encodes a putative mitochondrial HMG box-containing protein. In contrast to Abf2p, which contains two HMG boxes, Gcf1p contains only one C-terminal HMG box. In addition, it contains one putative coiled-coil domain with a potential role in protein dimerization. Fluorescence microscopy analysis of a C-terminally tagged Gcf1p with green fluorescent protein (GFP) revealed its mitochondrial localization in both heterologous (S. cerevisiae) and native (C. albicans) hosts. Biochemical analyses of DNA-binding properties indicate that Gcf1p is, similarly to Abf2p, a non-specific DNA-binding protein. To analyse the role of Gcf1p in mtDNA metabolism, we constructed strains lacking one functional allele of the GCF1 gene and carrying one GCF1 allele under the control of the MET3 promoter. Under repressible conditions this strain exhibited a more than 3000-fold decrease in levels of GCF1 mRNA, which was correlated with a substantial decrease in the number of mtDNA copies as well as recombination intermediates. The dramatic effect of reduced levels of Gcf1p on mtDNA metabolism indicates that the protein is involved in essential molecular transactions that relate to the mitochondrial genome.