Expression, functional characterization, and preliminary crystallization of the cochaperone prefoldin from the thermophilic fungus chaetomium thermophilum

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits, and has the appearance of a jellyfish: Its body consists of a double β-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. Based on structural information from archaeal prefoldins, mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the structure and mechanisms of eukaryotic prefoldins remain unknown. In this study, we succeeded in obtaining recombinant prefoldin from a thermophilic fungus, Chaetomium thermophilum (CtPFD). The recombinant CtPFD could not protect citrate synthase from thermal aggregation. However, CtPFD formed a complex with actin from chicken muscle and tubulin from porcine brain, suggesting substrate specificity. We succeeded in observing the complex formation of CtPFD and the group II chaperonin of C. thermophilum (CtCCT) by atomic force microscopy and electron microscopy. These interaction kinetics were analyzed by surface plasmon resonance using Biacore. Finally, we have shown the transfer of actin from CtPFD to CtCCT. The study of the folding pathway formed by CtPFD and CtCCT should provide important information on mechanisms of the eukaryotic prefoldin–chaperonin system.This research was supported by grants-in-aids for scientific research (JP18H04690, JP16H04572, JP16H00753 and JP15J08261) from the Ministry of Education, Science, Sports, and Culture of Japan, the Joint Studies Program (2017-2018) in the Okazaki BIO-NEXT project of National Institutes of Natural Sciences and a grant from CREST (JPMJCR13M1), the program of the Joint Usage/Research Center for Developmental Medicine (IMEG, Kumamoto University). This work was also supported by the grant BFU2016-75984 (AEI/FEDER, UE).Peer reviewe