Regulation of the Cytosolic Chaperonin Complex by Phosducin-like Protein

Brigham Young University, Provo UT

Investigators

Abstract

Eukaryotic cells have developed elaborate signal transduction pathways to respond to changes in their environment. The phosducin-family of proteins is believed to play a role in cellular signaling. This family consists of phosducin (Pd), which is expressed principally in the retina and pineal gland, and phosducin-like protein (PhLP), which is found in almost all tissues and cell types. Both Pd and PhLP bind to G proteins and other effectors. Pd and PhLP are regulators of G protein signaling pathways. Recent evidence suggests that Pd and PhLP also have other functions in the cell. Both have been found to interact with the SUG1 subunit of the 26S proteasome. Immunoprecipitation experiments and mass spectrometry identify proteins that co-immunoprecipitate with PhLP. These include subunits of the cytosolic chaperonin complex containing TCP1 1 (CCT). This chaperone is required for the folding of actin and tubulin and several other proteins. Thus, CCT is required for eukaryotic cell to produce essential protein products in their native state. PhLP binding inhibits the ability of CCT to fold these proteins. This research will elucidate the interaction of PhLP with CCT and investigate its physiological role in regulating CCT activity. The specific objectives are: 1) To test the hypothesis that PhLP is a physiological negative regulator of CCT activity; 2) To determine the mechanism of PhLP protein degradation and its role in regulating CCT activity; 3) To biochemically characterize the interaction between PhLP and CCT. The methodology includes a variety of biochemical and molecular biological techniques including gene silencing, analysis of site-directed mutants and chimeric proteins, cross-linking studies, immunological methods and mass-spectrometric approaches to elucidate PhLP functions and interactions. This study explores a totally novel function of PhLP and a unique mechanism that regulates CCT activity. Thus, it has the potential to forward our understanding of the regulation of protein folding in a major way as well as to elucidate the regulation and processing of proteins necessary for cell survival. This project also provides a research experience for undergraduates since a unique opportunity exists for undergraduate researchers to participate in this significant research project.