Eukaryotic prefoldin (PFD) is a heterohexameric chaperone with a jellyfish-like 
structure whose function is to deliver nonnative target proteins, principally 
actins and tubulins, to the eukaryotic cytosolic chaperonin for facilitated 
folding. Here we demonstrate that functional PFD can spontaneously assemble from 
its six constituent individual subunits (PFD1-PFD6), each expressed as a 
recombinant protein. Using engineered forms of PFD assembled in vitro, we show 
that the tips of the PFD tentacles are required to form binary complexes with 
authentic target proteins. We show that PFD uses the distal ends of different 
but overlapping sets of subunits to form stable binary complexes with different 
target proteins, namely actin and alpha- and beta-tubulin. We also present data 
that suggest a model for the order of these six subunits within the hexamer. Our 
data are consistent with the hypothesis that PFD, like the eukaryotic cytosolic 
chaperonin, has co-evolved specifically to facilitate the folding of its target 
proteins.