BIOCHEMICAL AND GENETIC ANALYSIS OF NUCLEAR TRANSPORT

许多核蛋白含有定位信号并在细胞核中积累

Many nuclear proteins contain localization signals and accumulate in the nucleus in an energy dependent fashion, however, the molecular basis for this process is unknown. We propose to further characterize a yeast nuclear envelope protein (p67) we have previously identified. p67 specifically recognizes nuclear localization sequences and is a good candidate for a receptor involved in the initiation of nuclear transport. p67 has been partially purified, and the gene encoding this protein has been isolated, and designated NSB1. Surprisingly, NSB1 contains two RNA-binding domains as well as an acidic N-terminus containing a number of serine clusters, and basic C-terminus. Anti-p67 antibodies stain the periphery of a substructure within the nucleus, possibly the nucleolus, by indirect immunofluorescence. The NSB1 protein either expressed under the GAL10 promoter or purified on a peptide affinity column, specifically recognizes the H2B nuclear localization sequence. The gene is being disrupted to test its essential nature. The role of NSB1 in nuclear transport in vivo will be studied in the following manner: Temperature-sensitive mutations will be isolated and characterized for defects in the nuclear transport pathway. Suppressors of the temperature-sensitive phenotype will be isolated and hopefully new proteins interacting with the NSB1 protein in the nuclear transport pathway will be identified. Where or how ATP is used during nuclear transport is unknown. Using the photoaffinity analog, 2-azido ATP, we have identified specific ATP-binding proteins at the nuclear envelope that may function during nuclear transport. Antibodies against the partially purified proteins are being used to clone the genes for these proteins.