Chaperone proteins bind dynamically to non-native proteins and assist them during their folding, refolding, translocation and degradation. We have discovered a novel hexameric chaperone, prefoldin, which binds to non-native proteins and delivers them to chaperonin, to which it also binds. We propose to investigate the role of prefoldin in protein metabolism in the cytosol of eukaryotes. I. We will investigate a) the target range of prefoldin and b) the state of bound target protein. c) We will study the assembly of prefoldin subunits in vitro and during translation. d) We will determine whether prefoldin (and/or chaperonin) act on target proteins cotranslationally or posttranslationally or both, and where most folding of their major substrates (actin and tubulin) occurs. e) We will study the geometry of chaperonin binding and prefoldin/target protein binding by electron microscopy. f) We will examine the role of prefoldin in heatshock. g) We will test the hypothesis that prefoldin affect the partitioning of newly synthesized proteins among the chaperones present in the cell. This will be done by performing in vitro experiments with potentially competing chaperones including hsp70s, and altering the balance among these chaperones in vivo. h) We will examine the effect of prefoldin on the ATPase activity of chaperonin. II. We propose to study the role of prefoldin homologs from the archaea, to elucidate the structure, function and evolutionary history of this highly conserved chaperone. We will coexpress the two prefoldin subunit homologs from the archaeon Methanococcus jannaschii in E. coli a) for structural analysis and b) to explore their interaction with the corresponding chaperonin as well as their effect on the folding by and polymerization of the corresponding archaeal chaperonin. These studies are designed to throw light on the workings of prefoldin and the evolution of chaperone proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059121-02
Application #
6181418
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Program Officer
Shapiro, Bert I
Project Start
1999-07-01
Project End
2003-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
2
Fiscal Year
2000
Total Cost
$264,285
Indirect Cost
Name
New York University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10016
Simons, C Torrey; Staes, An; Rommelaere, Heidi et al. (2004) Selective contribution of eukaryotic prefoldin subunits to actin and tubulin binding. J Biol Chem 279:4196-203