The proposed experiments are designed to identify the amino acid sequence that mediates the efflux of proteins from the nucleus. Rat hsc70, a well characterized member of the heat shock family that has been cloned and sequenced, will be used for this purpose. It has been demonstrated previously that this protein is capable of recycling between the nucleus and cytoplasm following injection into Xenopus oocytes, and that the rates of exchange across the envelope are similar to those observed for two homologous Xenopus polypeptides (B3 and B4). Deletion, and single amino acid mutagenesis of hsc70 will be performed in order to identify and characterize the efflux signal. In the deletion studies, fusion proteins (dihydrofolate reductase - hsc70) will be constructed to assure that the truncated forms of hsc70 do not cross the envelope by passive diffusion. The mutated forms of hsc70 and the hybrid constructs will be overexpressed in bacteria, labeled with 125I, and microinjected into the nucleus or cytoplasm of Xenopus oocytes. At various times after injection, transport across the nuclear envelope will be quantified by measuring radioactivity in manually isolated nuclear and cytoplasmic fractions.
NIH ExPorter
NIH ExPorter