CDNA CLONING AND EFFICIENT MITOCHONDRIAL IMPORT OF PRE-MTHSP70 FROM RAT-LIVER

Members of the 70-kD heat shock protein family have been found in all free-living organisms investigated and in major compartments of eukary otic cells where they are essential to a wide range of functions, incl uding protein folding and targeting, We have isolated a mitochondrial homolog (mtHSP70) from rat liver using ATP agarose affinity chromatogr aphy, Its identity was confirmed on the basis of immunological analysi s and Ca2+-dependent autophosphorylation. Using protein sequence obtai ned from the amino terminus and nine endo Lys-C peptide fragments, we have employed oligonucleotides to isolate a full-length cDNA clone, Th e open reading frame encodes a protein of 679 amino acids and calculat ed M(r) 73,913 daltons, The sequence has a high degree of identity wit h other members of the HSP70 family, including Escherichia call DnaK ( 51%), Sacharromyces cerevisiae SSC1p (65%), the constitutive cytosolic HSP70 from rat, HSC70 (46%), and the rat endoplasmic reticulum isofor m, BiP, (49%), The cDNA encodes a precursor protein with a 46-amino-ac id signal peptide that is absent from the protein isolated from rat li ver, The protein also shows a high degree of identity (98%) with a pro tein isolated from mouse and human tissues (PBP74, Domanico et al., 19 93; mortalin, Wadhwa et al., 1993a; CSA, Michikawa ed al., 1993a); how ever, the intracellular localization of these proteins is uncertain. W e show that the precursor of mtHSP70 is efficiently imported into isol ated mitochondria from rat liver and processed from 74 kD to the matur e 69-kD protein.