Molecular chaperones are required for protein folding under normal conditions in all cells and for responses to stress. Learning more about protein folding and the stress-response pathways will improve our understanding of many conditions, including Alzheimer's disease, Huntington's disease, and the survival of cancer cells. The ribosome-associated complex (RAC), consisting of the chaperones Zuo1 and Ssz1, has roles in both protein folding and signaling. RAC is perfectly position at the ribosomal exit tunnel to initiate proper folding of the nascent polypeptide chains as they are being translated by the ribosomes. In Saccharomyces cerevisiae, RAC activates the Hsp70 Ssb. After activation, Ssb binds to hydrophobic regions of the newly formed polypeptides to prevent protein aggregation. Only 1 molecule of RAC to every 50 ribosomes is required for normal growth, suggesting two possibilities: first, RAC may shuttle between ribosomes and help recruit factors to the ribosome, or second, RAC may be required to fold a subset of polypeptides on specialized ribosomes. Competition experiments in vitro and FRET experiments in vivo will determine whether RAC shuttles between ribosomes or if it is stably associated with ribosomes. If the latter is true, the specialized RAC-containing ribosomes will be purified and the associated mRNAs will be identified by microarray. Overexpression of RAC suggests a role for RAC away from the ribosome in the induction of pleiotropic drug resistance (PDR). During PDR, cells adapt to less than ideal conditions by facilitating an efflux of drugs out of the cell. PDR is similar to multidrug resistance, which occurs during the treatment of cancer cells. Little is known about the activation of PDR. To identify other factors required for the induction of PDR by RAC, a yeast deletion library will be screened. RAC signaling may couple the cell's translational status with the cell's stress-response pathways. Microarray analysis will be used to determine other downstream targets of RAC signaling. The mammalian homologs of RAC have been recently identified, so the results of these experiments may yield important insight into molecular chaperone function in higher eukaryotes and disease formation. PUBLIC HEALTH RELEVANCE Many diseases, which pose a significant health burden, are related to molecular chaperone dysfunction including Alzheimer's, Huntington's, and heart diseases. Molecular chaperones help cancer cells to survive and thus may be important therapeutic targets.