Kinetics and binding sites for interaction of the prefoldin with a group II chaperonin: contiguous non-native substrate and chaperonin binding sites in the archaeal prefoldin.
Prefoldin is a jellyfish-shaped hexameric co-chaperone of the group II chaperonins. It captures a protein folding intermediate and transfers it to a group II chaperonin for completion of folding. The manner in which prefoldin interacts with its substrates and cooperates with the chaperonin is poorly understood. In this study, we have examined the interaction between a prefoldin and a chaperonin from hyperthermophilic archaea by immunoprecipitation, single molecule observation, and surface plasmon resonance. We demonstrate that Pyrococcus prefoldin interacts most tightly with its cognate chaperonin, and vice versa, suggesting species specificity in the interaction. Using truncation mutants, we uncovered by kinetic analyses that this interaction is multivalent in nature, consistent with multiple binding sites between the two chaperones. We present evidence that both N- and C-terminal regions of the prefoldin beta sub-unit are important for molecular chaperone activity and for the interaction with a chaperonin. Our data are consistent with substrate and chaperonin binding sites on prefoldin that are different but in close proximity, which suggests a possible handover mechanism of prefoldin substrates to the chaperonin.
Okochi
Mina
M
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan.
Nomura
Tomoko
T
Zako
Tamotsu
T
Arakawa
Takatoshi
T
Iizuka
Ryo
R
Ueda
Hiroshi
H
Funatsu
Takashi
T
Leroux
Michel
M
Yohda
Masafumi
M
D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018833:Chaperonins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D019641:DNA, Archaeal / Q000235:genetics; D007700:Kinetics; D017014:Methanobacterium / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D017510:Protein Folding; D021122:Protein Subunits; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D018832:Molecular Chaperones; D021122:Protein Subunits; C112806:prefoldin; D018833:Chaperonins