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.