Purification and assay of the chaperone-dependent ubiquitin ligase of the carboxyl terminus of Hsc70-interacting protein.
It is notable that both chaperone and ubiquitin-proteasome systems are required for the removal of aberrant cellular proteins to ensure protein homeostasis in cells. However, the entity that links the two systems had remained elusive. The carboxyl terminus of Hsc70-interacting protein (CHIP), originally identified as a cochaperone of Hsc70, has both a TPR motif and a U-box domain. The TPR motif associates with Hsp70 and Hsp90, whereas the U-box domain executes ubiquitin ligase activity. Thus, CHIP is an ideal molecule, acting as a protein quality control ubiquitin ligase that selectively leads abnormal proteins recognized by molecular chaperones to degradation by the proteasome. This chapter describes methods of analyzing chaperone-dependent ubiquitin ligase activity of CHIP using firefly luciferase as a model substrate.
Murata
Shigeo
S
Laboratory of Frontier Science, The Tokyo Metropolitan Institute of Medical Science, Precursory Research for Embryonic Science and Technology (PRESTO), Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
Minami
Michiko
M
Minami
Yasufumi
Y
D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D048888:Fireflies / Q000201:enzymology; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins / Q000302:isolation & purification; D018841:HSP90 Heat-Shock Proteins / Q000302:isolation & purification; D006358:Hot Temperature; D006801:Humans; D049409:Luciferases, Firefly; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism*; D011489:Protein Denaturation; D013552:Swine; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000302:isolation & purification* / Q000378:metabolism*
D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D025801:Ubiquitin; D049409:Luciferases, Firefly; C492951:Stub1 protein, mouse; D044767:Ubiquitin-Protein Ligases