PBP74, A NEW MEMBER OF THE MAMMALIAN 70-KDA HEAT-SHOCK PROTEIN FAMILY, IS A MITOCHONDRIAL PROTEIN

The cloning of a cDNA encoding a new member of the highly conserved ma mmalian 70-kDa heat shock protein (hsp 70) family termed PBP74 was rec ently reported. Critical to an understanding of the function of this n ew hsp 70 is delineating its subcellular localization. Here we use a v ariety of immunological and biochemical approaches both in vitro and i n vivo to demonstrate that PBP74 is imported into and resides in mitoc hondria. By confocal immunofluorescence microscopy PBP74 is detected i n mitochondria, colocalizing with the mitochondrial 60-kDa heat shock protein. To address the inherent problem of serological cross-reactivi ty among the hsp70 family members, an influenza virus hemagglutinin ep itope tag was introduced into the PBP74 cDNA. The epitope-tagged PBP74 protein transiently expressed in L cells localized to mitochondria. M oreover, deletion of the N-terminal 46-amino acid presequence results in a cytosolic localization of the epitope-tagged protein. Cell fracti onation studies demonstrated PBP74 in purified mitochondria in a prote ase-protected location. After coupled transcription-translation the pr ecursor of PBP74 is imported into isolated yeast mitochondria, where i t becomes processed to the mature protein. According to a subfractiona tion of the mitochondria, the imported protein was found to be localiz ed in the matrix space. Import in vitro is time- and temperature-depen dent, requires matrix ATP, and is abolished upon depletion of the memb rane potential across the mitochondrial inner membrane. Similarly, in mammalian cells PBP74 is synthesized as a preprotein that requires mem brane,potential-dependent import into mitochondria for its maturation. Taken together, our data demonstrate that PBP74 is a mammalian mitoch ondrial hsp70.