Nascent actin and tubulin molecules undergo a series of complex interactions with chaperones and are thereby guided to their native conformation. These cytoskeletal proteins have the initial part of the pathway in common: both interact with prefoldin and with the cytosolic chaperonin containing tailless complex polypeptide 1. Little is understood with regard to how these chaperones and, in particular, prefoldin recognize the non-native forms of these target proteins. Using mutagenesis, we provide evidence that -actin and ␣-tubulin each have two prefoldin interaction sites. The most amino-terminally located site of both proteins shows striking sequence similarity, although these proteins are nonhomologous. Very similar motifs are present in -and ␥-tubulin and in the newly identified prefoldin target protein actin-related protein 1. Actin-related proteins 2 and 3 have related motifs, but these have altered charge properties. The latter two proteins do not bind prefoldin, although we identify them here as target proteins for the cytosolic chaperonin. Actin fragments containing the two prefoldin interaction regions compete efficiently with actin for prefoldin binding. In addition, they also compete with tubulins, suggesting that these target proteins contact similar prefoldin subunits.
Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other we study some of the molecular defects of three β-actin mutants that have been associated with diseases.
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