Preparation of a monoclonal antibody specific for a 48-kDa protein from mitochondrial nucleoids of the yeast, Saccharomyces cerevisiae.
Monoclonal antibodies (mAbs) were raised against yeast mitochondrial nucleoids (mt-nucleoids). In an analysis by a combination of immunofluorescence microscopy and staining with 4',6-diamidino-2-phenylindole (DAPI), one of them, designated YMN-1, distinctly stained mt-nucleoids, which were visible as dots, in spheroplasts and in isolated mitochondria. However, staining of isolated mt-nucleoids was rather weak. YMN-1 mAb recognized a 48-kDa protein in immunoblots of both mitochondrial and mt-nucleoid proteins. The 48-kDa protein was a minor component of mt-nucleoid proteins and was separated from extract of both mitochondria and mt-nucleoids by immunoaffinity chromatography. The affinity-purified 48-kDa protein reassociated with mt-nucleoids when mixed with isolated mt-nucleoids, as monitored by immunofluorescence microscopy. The results suggest that a large amount of 48-kDa protein is associated with mt-nucleoids in vivo, and that lysis of mitochondria by the treatment with detergent releases a considerable amount of this protein from mt-nucleoids during the isolation of mt-nucleoids.
Miyakawa
I
I
Biological Institute, Faculty of Science, Yamaguchi University, Japan.
Tokitaka
M
M
Shiiba
D
D
Sawada
T
T
Fujikura
Y
Y
Fukumoto
T
T
Sando
N
N
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D005656:Fungal Proteins / Q000032:analysis*; D005779:Immunodiffusion; D007211:Indoles; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008928:Mitochondria / Q000648:ultrastructure*; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000648:ultrastructure*; D013104:Spheroplasts / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D007211:Indoles; C007293:DAPI