Molecular cloning and characterization of a novel mitochondrial phosphoprotein, MIPP65, from rat liver.
A novel 65-kDa protein (designated MIPP65), which was phosphorylated by PKN in vitro in a manner highly dependent on arachidonic acid, was partially purified from the heat-stable proteins extracted from a 30,000g precipitate of rat liver. The cDNA clones were obtained by polymerase chain reaction using oligonucleotides based on partial amino acid sequences. The complete amino acid sequence deduced from the cDNAs contained two homologous regions with the mitochondrial NADH-ubiquinone oxidoreductase 9-kDa subunit precursor at the amino- and carboxyl-termini, whereas the central region was not related to any known proteins and contained a serine cluster. Northern blotting and immunoblotting analyses indicated that MIPP65 was expressed ubiquitously in rat tissues. Immunofluorescence analysis of the endogenous MIPP65 using polyclonal antiserum against MIPP65 showed a predominantly mitochondrial localization in C6 glioma cells. The recombinant MIPP65 expressed in COS7 cells showed a similar pattern of localization to that in C6 glioma cells. On the other hand, deletion of the amino-terminal region of MIPP65 abrogated such localization, indicating that the amino-terminal region contained a mitochondrial-targeting signal. From [32P]orthophosphate-labeled C6 glioma cells, the endogenous MIPP65 could be immunoprecipitated as a phosphoprotein with antiserum against MIPP65. These results suggest that MIPP65 is a novel mitochondrial phosphoprotein that is a candidate substrate for PKN.
Kitagawa
M
M
Faculty of Science, Kobe University, Kobe, 657, Japan.
Mukai
H
H
Ono
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D042967:Electron Transport Complex I; D005910:Glioma / Q000378:metabolism; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry; D009928:Organ Specificity; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D010710:Phosphates; D010750:Phosphoproteins; D011994:Recombinant Proteins; C108130:mitochondrial phosphoprotein, 65-kDa; D009247:NADH, NADPH Oxidoreductases; C061570:protein kinase N; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D042967:Electron Transport Complex I