The long term objective of this work is to understand the mechanisms by which large proteins are transported into the nucleus across the nuclear pore complex. Since import of most nuclear proteins occurs by selective mediated processes, the pore complex is likely to have an important role in controlling cell physiology. In this proposal, the specific aims are focused on detailed characterization of three proteins associated with the pore complex that have putative roles in protein import: the transport receptor for the nuclear location signal of the SV40 T antigen, the pore complex component that interacts with this transport receptor, and a major glycoprotein of the pore complex that contains O-linked N- acetylglucosamine. Monoclonal and domain-specific polyclonal antibodies will be prepared to the three proteins, and cDNA clones for the first two proteins will be isolated and sequenced. With these reagents, biochemical properties and localization of these proteins will be analyzed. Subsequently, the functions of these components will be investigated in cultures cells by microinjecting antibodies and overexpressing domains and deletion mutants of the proteins, to describe effects on mediated proteins import and on diffusional permeability of the pore complex. To complement this in vivo work, biochemical properties of nuclear proteins import assays. Using this system, antibodies to the three proteins considered above together with biochemical inhibitors will be used to begin dissecting the pathway for protein transport across the pore complex and to define intermediates in this process. Collectively, these studies will provide a paradigm for approaching study of RNA export from the nucleus, which probably shares many features with protein import.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM041955-01A2
Application #
3300456
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1990-04-01
Project End
1995-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
1
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
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
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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
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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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