Novel subfamily of mitochondrial HMG box-containing proteins: functional analysis of Gcf1p from Candida albicans.
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.
Visacka
Katarina
K
Departments of Genetics and Biochemistry, Faculty of Natural Sciences, Comenius University, 842 15 Bratislava, Slovakia.
Gerhold
Joachim M
JM
Department of General and Microbial Biochemistry, Institute of Molecular and Cell Biology, University of Tartu, Vanemuise 46-127, 51014 Tartu, Estonia.
Petrovicova
Jana
J
Departments of Genetics and Biochemistry, Faculty of Natural Sciences, Comenius University, 842 15 Bratislava, Slovakia.
Kinsky
Slavomir
S
Departments of Genetics and Biochemistry, Faculty of Natural Sciences, Comenius University, 842 15 Bratislava, Slovakia.
Jõers
Priit
P
Department of General and Microbial Biochemistry, Institute of Molecular and Cell Biology, University of Tartu, Vanemuise 46-127, 51014 Tartu, Estonia.
Nosek
Jozef
J
Departments of Genetics and Biochemistry, Faculty of Natural Sciences, Comenius University, 842 15 Bratislava, Slovakia.
Sedman
Juhan
J
Department of General and Microbial Biochemistry, Institute of Molecular and Cell Biology, University of Tartu, Vanemuise 46-127, 51014 Tartu, Estonia.
Tomaska
Lubomir
L
Departments of Genetics and Biochemistry, Faculty of Natural Sciences, Comenius University, 842 15 Bratislava, Slovakia.