Karyopherin-mediated protein transport across the nuclear pore complex (NPC) is vital for eukaryotic cells yet the mechanisms of karyopherin translocation across the NPC are unsolved. Tumor suppressor proteins, hormone receptors, and cell-cycle checkpoint control proteins are among the hundreds of essential regulatory proteins that need access across the NPC before executing their function in the nucleus. Thus, it is important to human health issues that we achieve a better understanding of the general mechanisms of nucleocytoplasmic transport. The long-term goal of this project is to understand at a molecular level how karyopherins use their interaction with nucleoporins to move across the NPC while carrying cargo. The experimental strategy is to use chemical crosslinkers to identify Nups that function as """"""""stepping stones"""""""" for Kap movement within the NPC, then to characterize in detail the interaction between karyopherins and identified nucleoporins using biochemical techniques, and finally to use the knowledge gained from biochemical analyses to design and conduct experiments that will test in vivo the mechanics of karyopherin movement within the NPC. The yeast S. cerevisiae will be used as a model eukaryote for this research.
The specific aims are: i) to test the hypothesis that nucleoporins containing FG repeats function as sequential """"""""stepping stones"""""""" in the movement of Kap95p across the NPC, ii) to test the hypothesis that nucleoporins are specifically arranged within the NPC to display a """"""""gradient of affinities"""""""" for Kap95p that promotes its movement across the NPC, iii) to identify point mutations in Kap95p that interfere with its ability to dock at distinct Nups and test their effects in vivo, and iv) to conduct a comparative study (as delineated for Kap95p) for two additional karyopherins (the exportin Crm1 p, and the importin Kapi 04p) with the goal of uncovering general and specific features in the paths of karyopherin movement in similar and opposite directions across the NPC.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061900-02
Application #
6644863
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Shapiro, Bert I
Project Start
2002-08-15
Project End
2007-07-31
Budget Start
2003-08-01
Budget End
2004-07-31
Support Year
2
Fiscal Year
2003
Total Cost
$281,139
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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Krishnan, V V; Lau, Edmond Y; Yamada, Justin et al. (2008) Intramolecular cohesion of coils mediated by phenylalanine--glycine motifs in the natively unfolded domain of a nucleoporin. PLoS Comput Biol 4:e1000145
Denning, Daniel P; Rexach, Michael F (2007) Rapid evolution exposes the boundaries of domain structure and function in natively unfolded FG nucleoporins. Mol Cell Proteomics 6:272-82
Pyhtila, Brook; Rexach, Michael (2003) A gradient of affinity for the karyopherin Kap95p along the yeast nuclear pore complex. J Biol Chem 278:42699-709