MOLECULAR-CLONING OF HUMAN P48, A TRANSIENT COMPONENT OF PROGESTERONE-RECEPTOR COMPLEXES AND AN HSP70-BINDING PROTEIN

A 48-kDa protein (p48) that transiently associates with progesterone r eceptor during cell-free assembly in rabbit reticulocyte lysate was is olated by two-dimensional gel separation. Tryptic peptide sequences we re generated and used to develop an antipeptide antiserum recognizing p48 by Western immunostaining, and this antiserum was used to monitor purification of native p48 from reticulocyte lysate. Eight mouse monoc lonal antibodies capable of immunoprecipitating vertebrate p48 were ge nerated. These monoclonal antibodies served as probes to clone ten p48 cDNAs from a HeLa cDNA expression library, One of the cloned cDNAs wa s sequenced in its entirety and codes for a 369-amino acid protein (ca lculated M(r) = 41,324), Northern blot analysis of RNA from multiple h uman tissues suggest that p48 may be ubiquitously expressed, Expressio n of human p48 cDNA in vitro yielded a product that comigrated with ra bbit p48 by SDS-PAGE and associated with progesterone receptor in a si milar manner, Immunoprecipitation of p48 complexes revealed a common a ssociation of p48 with hsp70 and, to a lesser extent, with hsp90 and p en, Thus, it appears that p48 is a novel component of the cytoplasmic molecular chaperone machinery.