This proposal focuses on the genetic and biochemical analysis of the yeast nuclear pore complex. The inhibits gained from this project and future studies spawned from it represent the development of a system to examine the molecular genetics of protein and RNA transport across the nuclear membrane. It is becoming increasingly apparent that many steps in embryogenesis are potentially regulated by post transcriptional events which may involve RNP transport through the nuclear pore. Further, the assembly events associated with the transition of hnRNP to mRNP at the nuclear pore have yet to be systematically examined. Preliminary studies indicate that the nuclear pore of yeast Saccharomyces cerevisiae exhibits an organization, structure, membrane density and fractionation properties which are similar to those described in other eukaryotes. Purification of a highly enriched yeast nuclear matrix pore lamina (MPL) fraction yields a protein profile with 12-15 proteins. Preliminary analysis of the MPL fraction indicate that some of its components are capable of forming intermediate like filaments in vitro and some proteins localize to the perinuclear region of the yeast nucleus. In the proposed studies antisera to yeast MPL proteins will be prepared and used to define localization in yeast and their coding DNA in lambda gtll libraries. These antisera will be coupled with an improved in vitro nuclear import assay to establish protein function relationships for nuclear transport. In vitro conditional alleles of MPL genes will be generated to establish structure- function relationships for selected components of the complex for binding and nuclear transport. Such characterized mutants would provide the starting points for more long term studies to establish the interaction and relationship of MPL components and the mechanism of translocation motors involved in transport through the nuclear envelope.
