CLONING AND SUBCELLULAR-LOCALIZATION OF HUMAN MITOCHONDRIAL HSP70

We report the cloning, nucleotide sequence, and localization of mitoch ondrial hsp70, a member of the human hsp70 multi-gene family, The huma n mthsp75 gene was cloned by screening an expression library with mono clonal antibody 3A3 that recognizes three members of the human hsp70 f amily (hsp70, hsc70, and a 75-kDa protein with characteristics identic al to that previously established for mitochondrial hsp70), The identi ty of the 75-kDa protein was confirmed by subcellular fractionation of HeLa cells and the demonstration that the 3A3-reactive 75-kDa protein co-fractionates with mitochondrial localized proteins, The nucleotide sequence of the respective cDNA clone revealed an open reading frame of 679 amino acids with extensive sequence identity with members of th e human hsp70 family, The derived amino-terminal pre-sequence shares f eatures common to other mitochondrial targeting sequences, The identit y of the cDNA was unequivocally established by introduction of an epit ope-tag at the carboxyl terminus of the cloned gene, transfection and analysis by immunofluorescence, The tagged 75-kDa protein localizes to mito chondria, thus providing conclusive evidence that it corresponds to the human mitochondrial hsp70, referred to here as mthsp75.