Macromolecular transport across the nuclear membrane plays a central role in the regulation of eukaryotic cell growth and development. The mechanism of nuclear transport is under investigation. Recently, nuclear localization sequences have been identified which selectively target proteins to the nucleus. An in vitro binding and import assay has been developed using isolated rat liver nuclei and peptide conjugates labelled with colloidal gold or with (125)I. Conjugates having the proper sequence bind to nuclei with a Kd=lOOnM. The nuclear membrane is a self assembling structure; when DNA is added to extracts of eggs of Xenopus laevis, nuclei form which resemble those of normal cells. The labelled conjugates enter these reformed nuclei. This system is being exploited to analyze the requirements for nuclear transport. The nuclear pore complex mediates transport across the nuclear membrane. The laboratory has demonstrated that proteins bearing cytoplasmically oriented, O-linked GlcNAc are components of the nuclear pore complex. To examine the function of these glycoproteins in nuclear transport, the CDNA encoding the major pore protein p62 was cloned. The gene encoding p62 is devoid of introns and has two potential transcription start sites. p62 has two domains: one myosin-like and one collagen-like. By transfecting an expression construct encoding p62 this protein has been overexpressed in cultured cells. Using the cloned CDNA it has also been demonstrated that 0-linked GlcNAc addition can be catalyzed in vitro in a rabbit reticulocyte lysate. Efforts are underway to identify the yeast homologue of p62 so that yeast genetics can be used to determine the normal function of p62. This will be combined with studies involving the addition of recombinant rat p62 to the Xenopus nuclear reconstitution assay.
