CLONING OF THE GENE ENCODING PEPTIDE-BINDING PROTEIN-74 SHOWS THAT ITIS A NEW MEMBER OF THE HEAT-SHOCK PROTEIN-70 FAMILY

We have previously described peptide-binding proteins of 72 and 74 kDa (PBP72/74), which have been implicated as playing a role in antigen p rocessing and are serologically related to the 70-kDa heat shock prote in (hsp70) family. Here we report the cloning and sequencing of the cD NA encoding PBP74 in mice and in humans, accomplished by using amino a cid sequence information obtained from the purified protein. We show t hat PBP74 is highly homologous to members of the hsp70 family but, sig nificantly, is not identical to any known member of this family. Inspe ction of the cDNA nucleotide sequence indicates that it encodes a 46-r esidue N-terminal peptide which is not present in the mature protein. Transcription and translation in vitro of the PBP74 cDNA verified that it encodes a form of PBP74 which is larger than the mature protein. T he presequence does not conform to known motifs for organelle-targetin g sequences, and at present, its function is not known. By confocal mi croscopy, PBP74 was localized to cytoplasmic vesicles but not to the n ucleus, mitochondria, or plasma membrane by using antibodies specific for the N-terminal 16 residues of PBP74. By RNA filter hybridization a nalysis, PBP74 mRNAs are detected in all cell types tested. Exposure o f cells to heat shock does not result in an increase in the mRNA level s of PBP74, unlike the dramatic increase observed for the stress-induc ible hsp70 mRNA. Thus, PBP74 appears to be a constitutive, new member of the hsp70 family.