Identification of the YMN-1 antigen protein and biochemical analyses of protein components in the mitochondrial nucleoid fraction of the yeast Saccharomyces cerevisiae.
We analyzed the protein components contained in the mitochondrial nucleoid (mt-nucleoid) fraction of the yeast Saccharomyces cerevisiae. Immunoblotting with anti-Abf2p antibody demonstrated the association of Abf2p, a major mitochondrial DNA-binding protein, with the mt-nucleoids. In contrast, porin and cytochrome c oxidase subunit III (CoxIIIp) were not detected by immunoblotting in the mt-nucleoid fraction. The YMN-1 monoclonal antibody recognized a 48 kDa protein of the mt-nucleoid fraction. The N-terminal amino acid sequence of the protein and immunological evidence showed that the YMN-1 monoclonal antibody recognizes dihydrolipoyl transsuccinylase (KE2), which is one of the constituents of the alpha-ketoglutarate dehydrogenase complex (KGDC). alpha-Ketoglutarate dehydrogenase (KE1) and dihydrolipoyl dehydrogenase (E3), which are other subunits of KGDC, were also detected in the mt-nucleoid fraction. An enzyme assay of the mt-nucleoid fraction showed that cytochrome c oxidase and fumarase activity were barely detected in the fraction, but the specific activity of KGDC in the mt-nucleoid fraction was relatively high and was approximately 60% of the specific activity in the mitochondrial fraction. Three components of KGDC were detected in the DNA-binding protein fractions after DNA-cellulose column chromatography of mt-nucleoid proteins. These results suggested that a part of KGDC in the mitochondrial matrix is associated with mt-nucleoids in vivo.
Sato
H
H
Department of Physics, Biology and Informatics, Faculty of Science, Yamaguchi University, Yamaguchi, 753-8512, Japan.
Tachifuji
A
A
Tamura
M
M
Miyakawa
I
I
D000217:Acyltransferases / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000946:Antigens, Fungal / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D015180:Electrophoresis, Gel, Two-Dimensional; D015151:Immunoblotting; D007655:Ketoglutarate Dehydrogenase Complex / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D008928:Mitochondria / Q000201:enzymology* / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology* / Q000276:immunology; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D000946:Antigens, Fungal; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D007655:Ketoglutarate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase; D000217:Acyltransferases; C021515:dihydrolipoamide succinyltransferase