The transport of large molecules between the nucleus and cytoplasm of eukaryotic cells occurs through large, multi-protein structures termed nuclear pore complexes (NPCs). Molecules being transported are targeted to and carried across the NPC while associated with members of a protein family termed the """"""""importins"""""""" or """"""""karyopherins"""""""" (Kaps). Different Kaps are responsible for the transport of distinct substrate molecules. While it is clear that the Kaps associate with proteins in the NPC during transport, it remains unclear exactly which Kaps bind which NPC proteins. In addition, the role this binding has in the physical translocation of the Kaps and their substrates across the NPC in unclear. In this proposal, the budding yeast Saccharomyces cerevisiae is used to examine the physical and functional interactions between the nuclear pore complex protein Nupip and all fourteen yeast karyopherins. In order to identify which Kaps have their kinetics of transport altered by a loss of Nupip, changes in the subcellular localization of Kaps expressed in NUP1 mutant cells will be examined by fluorescence microscopy using Kap::GFP fusions. Physical interactions between Nupip and each of the Kaps will also be explored using recombinant Nupip proteins and affinity column chromatography, as well as fluorescence light microscopy. Finally, a genetic screen used to identify mutants that suppress Nupi-mediated transport defects will be completed, and the resulting mutants examined for interactions with proteins in the karyopherin family. Importantly, original experiments examining Nupi-Kap interactions will be incorporated into an upper-level undergraduate Cell Biology laboratory course, as well as an intensive undergraduate research program, thus exposing large numbers of undergraduate students to the process of designing, performing, analyzing, and publishing basic research in molecular cell biology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15GM065107-01
Application #
6457210
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Shapiro, Bert I
Project Start
2001-07-01
Project End
2005-06-30
Budget Start
2001-07-01
Budget End
2005-06-30
Support Year
1
Fiscal Year
2001
Total Cost
$124,731
Indirect Cost
Name
Colgate University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
002253615
City
Hamilton
State
NY
Country
United States
Zip Code
13346
Finn, Erin M; DeRoo, Elise P; Clement, George W et al. (2013) A subset of FG-nucleoporins is necessary for efficient Msn5-mediated nuclear protein export. Biochim Biophys Acta 1833:1096-103
Belanger, Kenneth D; Griffith, Amanda L; Baker, Heather L et al. (2011) The karyopherin Kap95 and the C-termini of Rfa1, Rfa2, and Rfa3 are necessary for efficient nuclear import of functional RPA complex proteins in Saccharomyces cerevisiae. DNA Cell Biol 30:641-51
Belanger, Kenneth D (2009) Using affinity chromatography to investigate novel protein-protein interactions in an undergraduate cell and molecular biology lab course. CBE Life Sci Educ 8:214-25
Harper, Nicola C; Al-Greene, Nicole T; Basrai, Munira A et al. (2008) Mutations affecting spindle pole body and mitotic exit network function are synthetically lethal with a deletion of the nucleoporin NUP1 in S. cerevisiae. Curr Genet 53:95-105
Bembenek, Joshua; Kang, Jungseog; Kurischko, Cornelia et al. (2005) Crm1-mediated nuclear export of Cdc14 is required for the completion of cytokinesis in budding yeast. Cell Cycle 4:961-71
Belanger, Kenneth D; Gupta, Amitabha; MacDonald, Kristy M et al. (2005) Nuclear pore complex function in Saccharomyces cerevisiae is influenced by glycosylation of the transmembrane nucleoporin Pom152p. Genetics 171:935-47
Belanger, Kenneth D; Simmons, Laura A; Roth, Jessica K et al. (2004) The karyopherin Msn5/Kap142 requires Nup82 for nuclear export and performs a function distinct from translocation in RPA protein import. J Biol Chem 279:43530-9