Nucleocytoplasmic trafficking is essential for the biogenesis of the nucleus, for housekeeping functions such as transcription and ribosome assembly, and for the regulation of gene expression during the cell cycle, in development, and in response to changing environments. The nuclear pore complex is the site for the nucleocytoplasmic exchange of macromolecules and small metabolites and ions. The long term objectives of this proposal are to identify and characterize the cellular apparatuses that mediate major pathways of nuclear transport. This proposal is focused on analysis in yeast and vertebrate cells of nuclear export of the 40S ribosomal subunit, diffusion channels of the nuclear pore complex, and a putative nuclear import factor identified in a yeast genetics screen. Previous kinetic competition studies have shown that export of ribosomal subunits is saturable and utilizes specific export factors. Recently, using nuclear microinjection in Xenopus oocytes, the applicant has identified a portion of 18S rRNA that directs specific export.
Aim 1 proposes to delineate the sequences and associated proteins that specify 40S subunit export using biochemical and microinjection approaches in Xenopus oocytes and cultured cells. A complementary genetic approach will be pursued in yeast, beginning with the identification of 18S rRNA mutants defective in nuclear export.
Aim 2 will analyze the diffusive channels of the nuclear pore complex. A newly developed assay for diffusive export from yeast nuclei based on green fluorescent protein will be used to screen existing temperature and cold sensitive yeast mutant collections for mutations that cause defects in the function of the diffusion channels. In addition the potential regulation of diffusive channels in yeast and mammalian cells in different physiological states will be examined, and the size restrictions of the diffusive channel in yeast will be analyzed with NLS- GFP fusion proteins. A genetic selection in the applicant's laboratory recently identified a new yeast gene, NIP9, that has an apparent role in NLS- directed nuclear transport as well as protein translation.
Aim 3 proposes detailed analysis of the functions of the yeast NIP9 gene in protein synthesis and nuclear transport. This work will involve identification of Nip9p interacting proteins by biochemical and genetic means, mapping Nip9p domains required for ribosome binding and nuclear transport, and analysis of function in an in vitro transport and translation assays. In addition, the human Nip9p homologue will be cloned and functionally analyzed in cultured cells.
