Nucleophilic or N-proteins accumulate in the cell nucleus, many to nucleus/cytoplasm concentration ratios greater than 100; 1. Their distributions imply vital roles in controlling nuclear structure and gene activities. Eventual understanding of fundamental nuclear processes, including the modulation of gene expression during interphase, cell differentiation in development, and malignant transformation, will require elucidations of the mechanisms by which specific proteins concentrate in the nucleus, while the majority of the cell's proteins do not. Nuclear accumulation is a post-translational phenomenon, since N- proteins accumulate in the nucleus after they are isolated from cells (or synthesized in vitro) and microinjected into the cytoplasm. In several proteins, specific signal sequences (-5-20 amino acids long) have been identified which are necessary for accumulation. Nuclear accumulation mechanisms may include differential-affinity phase mechanisms (cytoplasmic exclusion or intranuclear binding) and/or nuclear envelope transport mechanisms (active transport or facilitated diffusion). But the separate contributions of these mechanisms have not been resolved for any single protein, largely because investigators have been unable to determine whether a protein is free (diffusive) or bound (nondiffusive) within the nucleus. We have developed methods which quantitatively distinguish free from bound proteins within the living cell, and we herein propose experiments to resolve and measure the mechanisms of nuclear protein accumulation in vivo. We will study the nucleocytoplasmic transport of (i) two widely-studied prototype N- proteins, nucleoplasmic and the simian virus 40 large T antigen (SV40 lg T), and (ii) macromolecular dextrans with covalently attached signal sequences of nucleoplasmic and SV40 lg T, i.e., signal-dextrans. We will microinject these molecules into the living amphibian oocyte, a cell especially suited for in vivo analysis of intracellular transport, and will use the internal reference phase, in situ nuclear envelope micropuncture, cryomicrodissection, and cryoautoradiography to measure the regional free and bound concentrations of the transported molecules (i) at steady-state (or equilibrium) and (ii) at times prior to steady-state, during periods of net flux across the nuclear envelope.
