The small heat shock protein (smHSP) and ␣-crystallin genes encode a family of 12-43-kDa proteins which assemble into large multimeric structures, function as chaperones by preventing protein aggregation, and contain a conserved region termed the ␣-crystallin domain. Here we report on the structural and functional characterization of Caenorhabditis elegans HSP16-2, a 16-kDa smHSP produced only under stress conditions. A combination of sedimentation velocity, size exclusion chromatography, and cross-linking analyses on wild-type HSP16-2 and five derivatives demonstrate that the Nterminal domain but not most of the the C-terminal extension which follows the ␣-crystallin domain is essential for the oligomerization of the smHSP into high molecular weight complexes. The N terminus of HSP16-2 is found to be buried within complexes which can accommodate at least an additional 4-kDa of heterologous sequence per subunit. Studies on the interaction of HSP16-2 with fluorescently-labeled and radiolabeled actin and tubulin reveal that this smHSP possesses a high affinity for unfolded intermediates which form early on the aggregation pathway, but has no apparent substrate specificity. Furthermore, both wild-type and C-terminally-truncated HSP16-2 can function as molecular chaperones by suppressing the thermally-induced aggregation of citrate synthase. Taken together, our data on HSP16-2 and a unique 12.6-kDa smHSP we have recently characterized demonstrate that multimerization is a prerequisite for the interaction of smHSPs with unfolded protein as well as for chaperone activity.Molecular chaperones belong to a class of proteins whose function is to interact with and stabilize proteins that are partially or totally unfolded, as is the case when proteins are in the process of being synthesized, translocated across a membrane, or damaged by conditions of cellular stress. Many chaperones are expressed at higher levels during biological stresses, and are members of heat shock protein (HSP) 1 families (1-4). Whereas some chaperones (HSP70, HSP40, and HSP60) are involved in protein folding under normal conditions in vivo (5-7), others such as HSP104 (8 -10), inducible HSP70s (11), and small HSPs (12-15) are known to play important roles in protecting organisms from stress.The small HSPs (smHSPs) form a structurally divergent protein family with members present in Archaea, Bacteria, and Eukarya (16, 17). The presence of an evolutionarily conserved ␣-crystallin domain distinguishes all smHSPs and ␣-crystallins (18 -20). This domain is preceded by an N-terminal domain, which is highly variable in size and sequence, and is followed by a short, poorly conserved C-terminal extension. Some smHSP genes contain an intron which delineates the N-terminal and ␣-crystallin domains (21, 22), and structural studies support a two-domain structure (20,23,24) consisting mostly of ␤-sheets (25, 26) for smHSPs. The C-terminal extensions of smHSPs appear relatively unstructured (27,28) and are known to undergo numerous modifications, including truncati...

Actin and tubulin polypeptide chains acquire their native conformation in the presence of the cytoplasmic chaperonin containing TCP-1 (CCT, also called TRiC) and, in the case of alpha- and beta-tubulin, additional protein cofactors. It has been previously demonstrated that nucleotide exchange and ATP hydrolysis act to switch CCT between conformations that interact either strongly or weakly with unfolded substrates [Melki, R., & Cowan, N.J. (1994) Mol. Cell. Biol. 14, 2895-2904]. The present study further documents the conformational changes and function of CCT. It is first shown, by the use of a range of labeled denatured substrate proteins and a radiolabeled total soluble HeLa cell extract, that CCT in the absence of nucleotides can bind any of a large number of proteins in vitro with high affinity. Second, by the use of denatured labeled beta-actin and beta-tubulin as model substrates for binding to CCT, we demonstrate that the CCT particle can contain two substrate protein chains simultaneously. Third, by electron microscopy, sedimentation velocity, and intrinsic fluorescence measurements, we document the conformational difference between CCT in its ATP- and ADP-bound forms, as well as the change that results from binding of substrate protein. A model summarizes substrate association with CCT and the role of the nucleotide in regulating the affinity of CCT for target proteins.

Small heat shock proteins (smHSPs) and ␣-crystallins constitute a family of related molecular chaperones that exhibit striking variability in size, ranging from 16 to 43 kDa. Structural studies on these proteins have been hampered by their tendency to form large, often dynamic and heterogeneous oligomeric complexes. Here we describe the structure and expression of HSP12.6, a member of a novel class of smHSPs from the nematode Caenorhabditis elegans. Like other members of its class, HSP12.6 possesses a conserved ␣-crystallin domain but has the shortest N-and C-terminal regions of any known smHSP. Expression of HSP12.6 is limited to the first larval stage of C. elegans and is not significantly upregulated by a wide range of stressors. Unlike other smHSPs, HSP12.6 does not form large oligomeric complexes in vivo. HSP12.6 was produced in Escherichia coli as a soluble protein and purified. Cross-linking and sedimentation velocity analyses indicate that the recombinant HSP12.6 is monomeric, making it an ideal candidate for structure determination. Interestingly, HSP12.6 does not function as a molecular chaperone in vitro, since it is unable to prevent the thermally induced aggregation of a test substrate. The structural and functional implications of these findings are discussed.

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CLIP-170 is a cytoplasmic linker protein that localizes to plus ends of microtubules in vivo. In this study, we have characterized the microtubule-binding properties of CLIP-170, to understand the mechanism of its plus end targeting. We show that the NH2-terminal microtubule-interacting domain of CLIP-170 alone localizes to microtubule plus ends when transfected into cells. Association of CLIP-170 with newly-formed microtubules was observed in cells microinjected with biotinylated tubulin, used as a tracer for growing microtubules. Using in vitro assays, association of CLIP-170 with recently polymerized tubulin is also seen. Cross-linking and sedimentation velocity experiments suggest association of CLIP-170 with nonpolymerized tubulin. We conclude from these experiments that the microtubule end targeting of CLIP-170 is closely linked to tubulin polymerization.