Isolation of the mitochondrial nucleoids from yeast Kluyveromyces lactis and analyses of the nucleoid proteins.
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.
Miyakawa
Isamu
I
Department of Physics, Informatics and Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan. miyakawa@po.cc.yamaguchi-u.ac.jp
Sato
Hiroshi
H
Maruyama
Yukari
Y
Nakaoka
Tomoko
T
D000595:Amino Acid Sequence; D004272:DNA, Mitochondrial / Q000737:chemistry*; D004278:DNA, Superhelical / Q000502:physiology; D004268:DNA-Binding Proteins / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007716:Kluyveromyces / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D024101:Mitochondrial Proteins / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D029701:Saccharomyces cerevisiae Proteins; D020539:Sequence Analysis, Protein; D013367:Submitochondrial Particles / Q000737:chemistry*; D014157:Transcription Factors / Q000302:isolation & purification
C072843:ABF2 protein, S cerevisiae; D004272:DNA, Mitochondrial; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors