No abstract

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.

Summary• Calcium (Ca 2+ ) signaling is thought to orchestrate responses to cellular stimuli. The efficacy of Ca 2+ signaling requires mediation by Ca 2+ -binding proteins.• The determination of the Arabidopsis genome sequence enables the identification of genes encoding potential Ca 2+ sensors.• Six Arabidopsis loci are defined as calmodulin ( CAM ) genes. Fifty additional genes are CAM-like ( CML ) genes, encoding proteins composed mostly of EF-hand Ca 2+ -binding motifs, have no other identifiable functional domains, and at least 16% identical with CaM. Number and structural diversity of the EF hands are evaluated. Intron/exon boundaries, phylogenetic tree and chromosomal distribution data for the CAMs and CMLs are presented.• Arabidopsis has 6 CAM genes, encoding only 3 isoforms. Maintenance of these genes suggests that they are unlikely to be fully redundant in function. Furthermore, the repeated EF hand motif is incorporated into at least 50 additional loci. The CaM relatives have altered EF hand number, organization, and predicted functional capacity. Additional structural differences and expression behaviors also indicate that the CML family has likely evolved distinct roles from the CAMs .

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.