Mechanisms regulating the nucleocytoplasmic distributions of proteins appear to be central to both normal and pathological cell differentiation. Understanding of these mechanisms has been limited by two technical problems. First, an inability to quantitatively localize specific diffusible macromolecules at the subcellular level and second, an inability to distinguish free from absorbed macromolecules in vivo. Recent technical advances, many from this laboratory, provide tools for the solution of both problems. We propose to use electrophoretic/chromatographic methods combined with ultra-low temperature microdissection and ultra-low temperature autoradiography to quantitatively localize specific endogenous polypeptides. In addition, we will use a newly-developed intracellular reference phase method to distinguish free and bound proteins. The study, to be performed in the amphibian oocyte, should lead to significant improvements in our knowledge of the mechanisms determining nucleo-cytoplasmic equilibria of proteins within living cells.
