Nuclear import is accomplished by nucleocytoplasmic shuttling receptors (importins) that transport cargo though the nuclear pore complex (NPC) by interaction with a series of nucleoporins. One of the critical regulators of import is the small GTPase Ran. The long-term goal of this work is to contribute to a detailed understanding of nucleocytoplasmic transport. This project is focused on molecularly characterizing how import complexes move through the NPC, and how this process is regulated by Ran.
The specific aims are: 1) The role of RanGTP in facilitating large cargo transport through the NPC will be analyzed in permeabilized cell assays using importin beta mutants deficient in nucleoporin binding and cargoes of varying size and import signal density. 2) The structure of a complex of RanGTP with full-length importin beta will be determined by X-ray crystallography. Importin beta mutants affecting Ran binding that are predicted to be deficient in cargo release will be examined. 3) The properties of a snurportin IBB/importin beta complex will be analyzed by biochemical and functional approaches. 4) The possibility that other import receptor complexes besides importin alpha/beta have a gradient of increasing affinity from the cytoplasmic-to-nuclear side of the NPC will be examined, and the functions of this gradient will be tested in permeabilized cell assays. The role of a nuclear-to-cytoplasmic nucleoporin affinity gradient in the recycling of importin beta/RanGTP complexes will be analyzed. It is hoped that this work will shed new light on some fundamental aspects of nuclear import mechanisms. Nucleocytoplasmic transport is central to the functioning of eukaryotic cells, and is an integral part of the processes that lead to most human diseases. Thus, understanding basic nuclear transport mechanisms can provide a framework for developing new therapeutic approaches relevant to viral pathogenesis, cancer and immunity and other processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041955-17
Application #
7046808
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Shapiro, Bert I
Project Start
1990-04-01
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2008-03-31
Support Year
17
Fiscal Year
2006
Total Cost
$458,223
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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Wang, Peng; Liu, Guang-Hui; Wu, Kaiyuan et al. (2009) Repression of classical nuclear export by S-nitrosylation of CRM1. J Cell Sci 122:3772-9
Ben-Efraim, Iris; Frosst, Phyllis D; Gerace, Larry (2009) Karyopherin binding interactions and nuclear import mechanism of nuclear pore complex protein Tpr. BMC Cell Biol 10:74
Cassany, Aurelia; Gerace, Larry (2009) Reconstitution of nuclear import in permeabilized cells. Methods Mol Biol 464:181-205
Ben-Efraim, Iris; Zhou, Quansheng; Wiedmer, Therese et al. (2004) Phospholipid scramblase 1 is imported into the nucleus by a receptor-mediated pathway and interacts with DNA. Biochemistry 43:3518-26
Koerner, Carolin; Guan, Tinglu; Gerace, Larry et al. (2003) Synergy of silent and hot spot mutations in importin beta reveals a dynamic mechanism for recognition of a nuclear localization signal. J Biol Chem 278:16216-21
Bednenko, Janna; Cingolani, Gino; Gerace, Larry (2003) Importin beta contains a COOH-terminal nucleoporin binding region important for nuclear transport. J Cell Biol 162:391-401
Bednenko, Janna; Cingolani, Gino; Gerace, Larry (2003) Nucleocytoplasmic transport: navigating the channel. Traffic 4:127-35
Wodrich, Harald; Guan, Tinglu; Cingolani, Gino et al. (2003) Switch from capsid protein import to adenovirus assembly by cleavage of nuclear transport signals. EMBO J 22:6245-55
Frosst, Phyllis; Guan, Tinglu; Subauste, Cecilia et al. (2002) Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export. J Cell Biol 156:617-30

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