Selective Contribution of Eukaryotic Prefoldin Subunits to Actin and Tubulin Binding

C Torrey Simons,An Staes,Heidi Rommelaere,Christophe Ampe,Sally A Lewis,Nicholas J Cowan

doi:10.1074/jbc.m306053200

C Torrey Simons, An Staes + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m306053200

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Abstract

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.

Highlights

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
The eukaryotic PFD hexamer is composed of six different subunits; these can be grouped into two ␣-like (PFD3 and -5) and four ␤-like (PFD1, -2, -4, and -6) subunits based on amino acid sequence similarity with their archaeal counterparts [23, 24]
The purified products behaved in a manner consistent with the size expected on the basis of the predicted open reading frame, with the exception of PFD3; this appeared smaller than the expected polypeptide

Summary

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Mercaptoethanol 0.1 M NaCl, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM DTT 20 mM Tris, pH 8.0, 1 mM EDTA, 2 mM DTT 20 mM Mes, pH 6.0, 1 mM EDTA, 1 mM DTT 1% Triton, 20 mM Tris, pH 8.6, 0.1 M NaCl, 5 mM EDTA, 2. MM DTT 8 M urea, 20 mM Tris, pH 8.6, 5 mM EDTA, 2 mM DTT 2 M urea, 20 mM Tris, pH 8.6, 0.2 M NaCl, 5 mM EDTA, 7. Mercaptoethanol 20 mM Tris, pH 8.6, 1 mM EDTA, 1 mM DTT 20 mM Tris, pH 8.0, 0.2 M NaCl, 1 mM EDTA, 1 mM DTT 20 mM Mes monohydrate, pH 6.8, 10 mM NaCl, 1 mM. These are growthcompromised and have characteristic cytoskeletal phenotypes [22, 23]. No authentic target proteins of any archaeal PFD have been identified. An important question concerns the contribution of each subunit to the binding of specific target proteins and the organization of the subunits within eukaryotic PFD. We show that the distal region of different but overlapping sets of subunits are critical for complex formation with actin versus ␣- and ␤-tubulin and suggest a model for the order of the six subunits within the heterohexamer

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