Isolation of the mitochondrial nucleoids from yeast Kluyveromyces lactis and analyses of the nucleoid proteins.

Abstract

Mitochondrial (mt) nucleoids were isolated from yeast Kluyveromyces lactis with morphological intactness. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed more than 20 proteins that are associated with the mt-nucleoids. However, the protein profile of the mt-nucleoids of K. lactis was significantly different from that of the mt-nucleoid proteins from Saccharomyces cerevisiae. SDS-DNA PAGE, which detected an Abf2p, a major mitochondrial DNA-binding protein, among the mt-nucleoid proteins of S. cerevisiae on a gel, detected only a 17-kDa protein in the K. lactis mt-nucleoid proteins. The 17-kDa protein was purified as homogeneous from the mt-nucleoids by a combination of acid extraction, hydroxyapatite chromatography and DNA-cellulose chromatography. The 17-kDa protein introduced a negative supercoil into circular plasmid DNA in the presence of topoisomerase I, as does S. cerevisiae Abf2p, and it packed K. lactis mtDNA into nucleoid-like particles in vitro. These results, together with the determination of the N-terminal amino acid sequence, suggested that the 17-kDa protein is an Abf2p homologue of K. lactis and plays structural roles in compacting mtDNA in cooperation with other nucleoid proteins.