CLONING AND SOME NOVEL CHARACTERISTICS OF MITOCHONDRIAL HSP70 FROM CHINESE-HAMSTER CELLS

The cDNA for Chinese hamster mitochondrial Hsp70 (mHsp70) was cloned a nd sequenced using a polymerase chain reaction probe based on conserve d regions in the Hsp70 family of proteins. The encoded protein consist s of 679 amino acids which includes a N-terminal mitochondrial targeti ng sequence of 46 amino acids. The mHsp70 protein contains several seq uence signatures that are characteristics of prokaryotic and eukaryoti c organellar Hsp70 homologs. In a phylogenetic tree based on Hsp70 seq uences, it branches with the gram-negative proteobacteria, supporting the endosymbiotic origin of mitochondria from this group of prokaryote s. The mHsp70 cDNA was transcribed and translated in vitro and its imp ort into isolated rat heart mitochondria was examined. The precursor m Hsp70 was converted into a mature form of lower molecular mass (approx imate to 71 kDa) which became resistant to trypsin digestion. The impo rt of mHsp70 into mitochondria was not observed in the presence of an uncoupler of energy metabolism or when the N-terminal presequence was lacking. The cDNA for mHsp70 was expressed in Escherichia coil and a p olyclonal antibody to the purified recombinant protein was raised. The antibody shows no cross-reactivity to recombinant cytosolic Hsp70 pro tein and in 2-D gel blots it reacted specifically with the mHsp70 prot ein only. In immunofluorescence experiments, the antibody predominantl y labeled mitochondria, and the observed labeling pattern was identica l to that seen with a monoclonal antibody to the mitochondrial Hsp60 c haperonin. The affinity-purified antibody to mHsp70 was also employed to examine the subcellular distribution of the protein by cryoelectron microscopy and the immunogold-labeling technique. In these experiment s, in addition to mitochondria, labeling with mitochondrial Hsp70 anti body was also observed on the plasma membrane and in unidentified cyto plasmic vesicles and granules. These studies raise the possibility tha t similar to the Hsp60 chaperonin and a number of other mitochondrial proteins, mHsp70 may have an extramitochondrial role. (C) 1997 Academi c Press.