NUCLEOLAR-CYTOPLASMIC TRANSPORT

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Exchange of macromolecules between the nucleolus and the cytoplasm is an essential process of all eukaryotic cells. We previously identified Nopp140, a nuclear localization signal binding protein that shuttles between the nucleolus and the cytoplasm on highly localized tracks. The long-term objective of this giant proposal is to understand the mechanism and regulation of Nopp140 mediated nucleolar-cytoplasmic transport. Specifically: 1. Characterization of NAP57 and identification of additional Nopp140 associated proteins. We have identified a Nopp140 associated protein, NAP57, in rat liver nuclear extracts that localized to Nopp140 like intranuclear tracks, and cloned its cDNA. We propose to: (a) Determine if NAP57 also shuttles between nucleus and cytoplasm. (b) Study the interaction of endogenous and bacterially expressed NAP57 with Nopp140. (c) Identify additional Nopp140 interacting proteins by affinity chromatography of cellular fractions using columns of recombinant Nopp140 and NAO57. 2. Structure and dynamics of Nopp140 tracks. To develop a system that allows an easier detection of the tracks and that will enable us to simultaneously manipulate cellular incubation conditions, we will visualize them by laser scanning confocal microscopy and three dimensional reconstruction of optical sections of: (a) immunolocalized Nopp140 and NAP57, and (b) fluorescently labeled recombinant Nopp140 and Nap57, after microinjection or in in vitro nuclear import assays. 3. Phosphorylation of Nopp140. We previously showed that Nopp140 is one of the most highly phosphorylated proteins in the cell and that nuclear localization signal binding is dependent on its phosphorylation state. By employing subcellular fractionation and pulse chase labeling techniques, it will be determined if and how the phosphorylation state of Nopp140 varies according to its subcellular location. Since we have shown that recombinant Nopp140 can serve as kinase substrate in vitro, we will exploit it to identify and purify the cellular Nopp140 kinase(s). 4. Identification of the Nopp140 homolog in yeast. Preliminary data show that the rat Nopp140 cDNA cross- hybridizes with a specific yeast mRNA, and that polyclonal antibodies raised against the mammalian protein crossreact with yeast nucleolar proteins. Based on these homologies, we will clone and sequence the yeast Nopp140 gene and obtain further insight into its function by: (a) Studying the effects of gene disruption. (b) Analyzing its subcellular location and phosphorylation state in mutant strains. (c) Exploiting molecular genetic techniques. Ultimately, these studies will contribute to the understanding of the upregulation of ribosome synthesis and the resulting nucleolar hyperactivity which are major characteristics of every cancer cell. In fact, the importance of the nucleolus in the regulation of cell growth has been emphasized by the recent discovery of the nucleolar oncoprotein LYAR.