Chaperonins assist the folding of other proteins. Type II chaperonins, such as chaperonin containing TCP-1(CCT), are found in archaea and in the eukaryotic cytosol. They are hexadecameric or nonadecameric oligomers composed of one to eight different polypeptides. Whereas type I chaperonins like GroEL are promiscuous, assisting in the folding of many other proteins, only a small number of proteins, mainly actin and tubulin, have been described as natural substrates of CCT. This specificity may be related to the divergence of the eight CCT subunits. Here we have obtained a three-dimensional reconstruction of the complex between CCT and alpha-actin by cryo-electron microscopy and image processing. This shows that alpha-actin interacts with the apical domains of either of two CCT subunits. Immunolabelling of CCT-substrate complexes with antibodies against two specific CCT subunits showed that actin binds to CCT using two specific and distinct interactions: the small domain of actin binds to CCTdelta and the large domain to CCTbeta or CCTepsilon (both in position 1,4 with respect to delta). These results indicate that the binding of actin to CCT is both subunit-specific and geometry-dependent. Thus, the substrate recognition mechanism of eukaryotic CCT may differ from that of prokaryotic GroEL.

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Three-dimensional reconstruction from cryoelectron micrographs of the eukaryotic cytosolic chaperonin CCT complexed to tubulin shows that CCT interacts with tubulin (both the a and b isoforms) using ®ve speci®c CCT subunits. The CCT±tubulin interaction has a different geometry to the CCT±actin interaction, and a mixture of shared and unique CCT subunits is used in binding the two substrates. Docking of the atomic structures of both actin and tubulin to their CCT-bound conformation suggests a common mode of chaperonin±substrate interaction. CCT stabilizes quasi-native structures in both proteins that are open through their domain-connecting hinge regions, suggesting a novel mechanism and function of CCT in assisted protein folding. Keywords: actin/chaperonin/electron microscopy/protein folding/tubulin IntroductionFolding of many proteins in vivo requires interaction with macromolecular complexes known as chaperonins. These proteins are ubiquitous oligomeric assemblies that have been classi®ed into two distinct families that share limited but signi®cant sequence homology: type I, present in eubacteria and endosymbiotic organelles, and of which the bacterial GroEL is the best known representative; and type II, present in archaebacteria and the eukaryotic cytosol, which are represented by the thermosome and CCT (chaperonin containing TCP-1), respectively (Bukau and Horwich, 1998;Gutsche et al., 1999;Willison, 1999). Most of the chaperonins share a common architecture, a cylinder made up of two back-to-back stacked rings, each one enclosing a cavity where folding takes place. The atomic structures of GroEL (Braig et al., 1994) and the type II thermosome (Ditzel et al., 1998) have revealed a common subunit architecture consisting of three domains: apical, intermediate and equatorial. The equatorial domain provides most of the intra-and inter-ring interactions and contains the binding site for ATP, the hydrolysis of which is necessary for the working cycle of the chaperonin, while the apical domain is involved in substrate binding and undergoes large conformational changes during the folding cycle. There are, however, numerous differences between type I and type II chaperonins, one of which is the absence of co-chaperonins for type II family members, whose role in the closure of the cavity during the chaperonin working cycle is ful®lled instead by a helical protrusion in the apical domain (Klumpp et al., 1997;Ditzel et al., 1998;Llorca et al., 1999a). Another important difference is related to the degree of complexity of the chaperonin ring, ranging from the seven identical subunits of type I chaperonin GroEL to eight different polypeptide subunits in the case of the type II chaperonin CCT. The most important difference between these two chaperonins is, however, related to their substrate speci®city: whereas GroEL interacts with a broad range of substrates (Houry et al., 1999) using a non-speci®c recognition mechanism based on hydrophobic interactions (Bukau and Horwich, 1998;Chen and Sigler, 1999;Shtilerman et al., 19...

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets specific cell cycle-related proteins for degradation, regulating progression from metaphase to anaphase and exit from mitosis. The APC is regulated by binding of the coactivator proteins Cdc20p and Cdh1p, and by phosphorylation. We have developed a purification strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC-associated proteins, Swm1p and Mnd2p. Using an in vitro ubiquitylation system and a native gel binding assay, we have characterized the properties of wild-type and mutant APC. We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding. APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities. However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a specific role in substrate recognition by APC-coactivator complexes. These results imply that Doc1p/Apc10 may play a role to regulate the binding of specific substrates, similar to that of the coactivators.