Kinetics and Binding Sites for Interaction of the Prefoldin with a Group II Chaperonin

Abstract

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 ␤ subunit 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.Molecular chaperones are ubiquitous proteins that are required for the correct folding, assembly, transport, and degradation of proteins within the cell (1). One class of chaperones, termed chaperonins, are seven-to nine-membered double ring complexes of 800 -1000 kDa that capture non-native proteins in their central cavity to promote correct folding in an ATP-dependent manner (2-5). They are classified into two groups, group I found in bacteria and organelles of eukaryotes, and group II in archaea and in the cytoplasm of eukaryotes (6, 7). The bacterial group I chaperonin, GroEL, is a cylinder-shaped tetradecamer that is capped by the heptameric co-chaperone, GroES (8 -10). In contrast, the group II chaperonin exists as an eight-or nine-rotationally symmetric double ring in a toroidal structure composed of homologous subunits of about 60 kDa and functions independently of a co-chaperone corresponding to GroES (11,12). The crystal structures of the group II chaperonins from the acidothermophilic archaeum Thermoplasma acidophilum and the hyperthermophilic archaeum Thermococcus sp. strain KS-1 suggest that the long helical protrusions located at the opening of their cavities, in their apical domains, play the equivalent role of GroES as a built-in lid of the cavity (13-15). The archaeal chaperonin takes an open conformation in the nucleotide-free or ADP-bound states and changes to a closed conformation upon binding ATP (16,17). Interestingly, closure of the built-in lid of CCT, 1 the eukaryotic cytosol group II chaperonin, is induced not by the binding but by the hydrolysis of ATP (18).The group II chaperonins cooperate with a co-chaperone, prefoldin/GimC (19 -21). Prefoldin (PFD) has been shown to participate in the maturation of actin and members of the tubulin family by transferring them in the incomplete...

Localization of Prefoldin Interaction Sites in the Hyperthermophilic Group II Chaperonin and Correlations between Binding Rate and Protein Transfer Rate

“…4 The subunit composition of archaeal prefoldin consists of α 2 β 4, whereas eukaryotic prefoldins are composed of six different subunits (two α-type and four β-type subunits). Archaeal prefoldins from Methanobacterium thermoautotrophicum (MtGimC) [5][6][7] and Pyrococcus horikoshii OT3 (PhPFD) 6,[8][9][10][11] have been characterized in detail. They are capable of stabilizing non-native proteins and releasing them for subsequent chaperonindependent folding in vitro.…”

The HCV ARFP/F/core+1 protein: Production and functional analysis of an unconventional viral product

“…This interaction disturbed the normal function of the PFD complex, which is to interact with nascent polypeptide chains to stabilize nonnative proteins, such as actins and tubulins, for subsequent folding in the central cavity of a chaperonin (102,103). The core11/F-PFD2 interaction, first observed in the yeast two-hybrid system and then confirmed by coimmunoprecipitation analysis and confocal microscopy in human embryonic kidney (HEK293) cells, resulted in an aberrant organization of the tubulin cytoskeleton, with a low density of microtubules in the perinuclear area (possibly due to the prevention of growth of the microtubules minus ends at the organizing centers).…”

Single molecule FRET detection in CdSe-QD donor and Cy5-labeled molecular chaperone acceptor complex by imaging microscopy

“…We use prefoldin (PFD, Pyrococcus horikoshii OT3, 86 kDa) [10] in this experiment. Our protocol for the conjugation in Hepes buffer solution of pH 7.5 with or without potassium chloride (HK) is in Ref.…”