The paradigm sirtuin, Sir2p, of budding yeast is required for establishing cellular age asymmetry, which includes the retention of damaged and aggregated proteins in mother cells. By establishing the global genetic interaction network of SIR2 we identified the polarisome, the formin Bni1p, and myosin motor protein Myo2p as essential components of the machinery segregating protein aggregates during mitotic cytokinesis. Moreover, we found that daughter cells can clear themselves of damage by a polarisome-and tropomyosin-dependent polarized flow of aggregates into the mother cell compartment. The role of Sir2p in cytoskeletal functions and polarity is linked to the CCT chaperonin in sir2D cells being compromised in folding actin. We discuss the findings in view of recent models hypothesizing that polarity may have evolved to avoid clonal senescence by establishing an aging (soma-like) and rejuvenated (germ-like) lineage.

The levels of oxidatively damaged, carbonylated, proteins increase with the replicative age of yeast mother cells. We show here that such carbonylated proteins are associated with Hsp104p-containing protein aggregates and that these aggregates, like oxidized proteins, are retained in the progenitor cell during cytokinesis by a Sir2p-dependent process. Deletion of HSP104 resulted in a breakdown of damage asymmetry, and overproduction of Hsp104p partially restored damage retention in sir2⌬ cells, suggesting that functional chaperones associated with protein aggregates are required for the establishment of damage asymmetry and that these functions are limited in sir2⌬ cells. In line with this, Hsp104p and several Hsp70s displayed elevated damaged in sir2⌬ cells, and protein aggregates were rescued at a slower rate in this mutant. Moreover, overproduction of Hsp104p suppressed the accelerated aging of cells lacking Sir2p, and drugs inhibiting damage segregation further demonstrated that spatial quality control is required to rejuvenate the progeny.

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.

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...

Folding to completion of actin and tubulin in the eukaryotic cytosol requires their interaction with cytosolic chaperonin CCT [chaperonin containing tailless complex polypeptide 1 (TCP-1)]. Three-dimensional reconstructions of nucleotide-free CCT complexed to either actin or tubulin show that CCT stabilizes both cytoskeletal proteins in open and quasi-folded conformations mediated through interactions that are both subunit speci®c and geometry dependent. Here we ®nd that upon ATP binding, mimicked by the non-hydrolysable analog AMP-PNP (5¢-adenylylimido-diphosphate), to both CCT±a-actin and CCT± b-tubulin complexes, the chaperonin component undergoes concerted movements of the apical domains, resulting in the cavity being closed off by the helical protrusions of the eight apical domains. However, in contrast to the GroE system, generation of this closed state does not induce the release of the substrate into the chaperonin cavity, and both cytoskeletal proteins remain bound to the chaperonin apical domains. Docking of the AMP-PNP±CCT-bound conformations of a-actin and b-tubulin to their respective native atomic structures suggests that both proteins have progressed towards their native states.