Molecular chaperones play an important part in a number of fundamental cellular processes. This includes a role for chaperones in the initial folding of nascent polypeptide chains as they are being synthesized and released from the ribosome. Likewise, chaperones play an important role in the trafficking of proteins throughout the cell and into various organelles. One interest of our lab concerns the role that chaperones play in the trafficking of proteins that are targeted to degradation pathways. We have recently shown that the yeast molecular chaperone Ssa2p plays an important role in the degradation of fructose-1,6-bisphosphatase (FBPase) a key gluconeogenic enzyme. FBPase is targeted to the vacuole for degradation when glucose starved cells are fed glucose. The trafficking of this enzyme to the vacuole requires at least two steps. The first involves the import of FBPase into intermediate transport vesicles (Vid vesicles), while in the second step, the Vid vesicles traffick to the vacuole, where FBPase is degraded. In the absence of Ssa2p, FBPase remains trapped in the cytosol and cannot traffick into the vacuole. Via the use of an in vitro assay, we have identified the site of action of this defect as the Vid vesicles. In the absence of Ssa2p, FBPase import into Vid vesicles is blocked and the protein cannot be delivered to the vacuole. However, the re-addition of purified recombinant protein restored the ability of these vesicles to import FBPase. Thus we have identified a novel role for chaperones in the trafficking of proteins into cellular vesicles. Our ongoing research involves the identification and characterization of other proteins that are required for the trafficking and degradation of FBPase. At present, a number of novel as well as previously characterized proteins have been identified through genetic and biochemical screenings. These include other members of molecular chaperone families, proteins involved in the ubiquitination process, and proteins with no other ascribed function.

Another interest of our lab concerns the misfolding of genetically mutated proteins. There are a number of medically relevant protein folding mutations including those associated with the CFTR gene (mutated in cystic fibrosis) and the p53 gene (often mutated in cancer). For these diseases, either deletion or missense mutations result in the production of a protein that cannot assume its properly folded conformation. Accordingly, the proteins are either rapidly degraded or sequestered in an improper cellular location, resulting in their inactivation. One of our research interests is to determine what role molecular chaperones played in this process and whether manipulation of chaperone levels may influence the folding of these proteins. In a similar manner, we have attempted to manipulate the protein folding environment inside the cell in such a way as to induce the proper folding of proteins that would otherwise misfold. These experiments involve the use of protein stabilizing agents that we refer to as "chemical chaperones". We found that treatment of cells with the chemical chaperone glycerol resulted in the proper folding and processing of the mutant delta F508 CFTR protein. Importantly, these treatments induced the movement of the delta F508 CFTR protein to the plasma membrane, where it formed a functional chloride channel. In other studies, similar treatments were effective in inducing the proper folding, localization and function of a mutant form of p53. Therefore, an ongoing goal of our work is to determine the mode of action of chemical chaperones and to identify other compounds that are effective in correcting protein folding defects in living cells.