Hsp70 co-factors in budding yeast include two disaggregases, the AAA+ protein Hsp104, related to bacterial ClpB, and the nucleotide exchange factor Sse1, a member of the Hsp110 family. We have used single particle electron microscopy (EM) and three-dimensional (3D) reconstruction to study the structure and action of the ClpB ATPase machinery. Hsp104 and ClpB are members of an Hsp100 subfamily specific for disaggregation of aggregates, and they contain a coiled-coil propeller (middle domain) inserted near the end of the first ATPase domain. Using the BAP variant of ClpB that forms complexes with the protease ClpP, we obtained 3D EM maps that revealed head-to-tail contacts between adjacent middle domains. Some of these contacts must be released in order for Hsp70 binding and activation.

In related work, we are using electron tomography to study the 3D arrangement of prion aggregates in yeast cells, in particular to examine the roles of the disaggregases in the organization of cellular aggregates. [PSI+] prions are deposited as regular bundles of fibrils in distinct, but not membrane-bound, assemblies in the yeast cytoplasm. The aggregates are thought to result from a balance between polymerization of amyloid forming regions and breakdown by disaggregases, a process that also results in transmission of the [PSI+] phenotype to other cells. We observe changes in the structure and arrangement of the fibrils in yeast cells when components of the Hsp70 system are depleted or over-expressed. The findings support the idea that Hsp70 and its co-factors play a major role in determining the prion fibril structure and organization in yeast cells.