Abstract: Molecules of mitochondrial DNA (mtDNA) are packed into nucleo-protein complexes termed mitochondrial nucleoids (mt-nucleoids). In this study, we analyzed mt-nucleoids of the yeast Candida parapsilosis, which harbors a linear form of the mitochondrial genome. To identify conserved as well as specific features of mt-nucleoids in this species, we employed two strategies for analysis of their components. First, we investigated protein composition of mt-nucleoids isolated from C. parapsilosis mitochondria, determined N-terminal amino acid sequences of fourteen proteins associated with the mt-nucleoids and identified corresponding genes. Next, we complemented the list of mt-nucleoid components by additional candidates identified in the complete genome sequence of C. parapsilosis as homologs of Saccharomyces cerevisiae mt-nucleoid proteins. Our approach revealed several known mt-nucleoid proteins as well as additional components expanding the repertoire of proteins associated with these cytological structures. Particularly, we identified and purified the protein Gcf1, which is abundant in the mt-nucleoids and exhibits common structural features with the mtDNA packaging protein Abf2 from S. cerevisiae. We demonstrate that Gcf1p co-localizes with mtDNA, has DNA-binding activity in vitro, and is able to stabilize mtDNA in S. cerevisiae Deltaabf2 mutant, which points to its role in the maintenance of C. parapsilosis mitochondrial genome. Importantly, in contrast to Abf2p, in silico analysis of Gcf1p predicted the presence of a coiled-coil domain and a single high-mobility group (HMG) box, suggesting that it represents a novel type of mitochondrial HMG protein.