The proper compartmentalization of proteins destined for the cell nucleus is likely to play a role in the regulation of cell growth and development. Synthetic peptides containing the amino acid sequence responsible for the nuclear localization of the SV40 Large T antigen and a modified sequence present in a cytoplasmic variant have been used to study protein import into the nucleus. These synthetic peptides have been used to generate polyclonal and monoclonal antibodies which bind specifically to the nuclear localization sequence. Such short peptides, when chemically coupled to the large fluorescent protein beta-phycoerythrin, specifically target the protein conjugate to the nucleus. Transport of such conjugates across the nuclear envelope was demonstrated after micro-infection into cultured cells or in an in vitro import assay using rat liver nuclei. Transport is time, temperature and energy dependent; only conjugates containing the localization sequence are properly transported. The nuclear pore complex transverses the nuclear envelope and may mediate uptake int0 the nucleus. We have shown that the outer nuclear membrane is an important site of membrane glycoprotein synthesis. We have also demonstrated that proteins bearing cytoplasmically oriented, O-linked GlcNAc are components of the nuclear pore complex. The nuclear pore glycoproteins can be selectively labelled using the lectin wheat germ agglutinin. This lectin reversibly blocks import into the nucleus. Monoclonal antibodies have been raised against these nuclear pore glycoproteins and O-linked GlcNAc was found to be part of the immunodeterminant. These findings raise the exciting possibility that cytoplasmic glycosylation may be involved in the assembly or functioning of the nuclear pore.
