]: Misfolded and unassembled proteins are retained in the endoplasmic reticulum via a process termed """"""""quality control,"""""""" ensuring that only native proteins are secreted to the extracellular space. Quality control plays a central role in diseases such as cystic fibrosis and alpha1-antitrypsin deficiency, and also determines the yield of eucaryotic expression systems for production of pharmaceutical proteins. The long term goal of the proposed research is to gain a detailed biophysical understanding of the quality control process, with the specific aims of examining: the kinetic competition between folding, unfolding, retention, and ER export; and the roles of the oxidase, disulfide isomerase, chaperone, and anti-chaperone activities of the foldase PDf in these kinetic processes. The experimental system for these studies is bovine pancreatic trypsin inhibitor (BPTI) expressed in the yeast Saccharomyces cerevisiae. 29 mutants of BPTI varying in stability, number of cysteines, and surface charge distribution have been constructed and secreted from yeast. A pulse-chase radiolabeling protocol has been developed which allows measurement of the rates of folding, ER export, Golgi-surface export, and degradation. Correlations between secretion efficiency and biophysical attributes of the BPTI mutants indicate that stability, surface charge distribution, presence of free thiols, or the deletion of disulfides can each strongly influence the efficiency of escape from ER quality control. The quantitative interaction of these attributes of BPTI with the ER quality control apparatus will be examined by determining the intracellular rates of folding, ER export, and degradation for a subset of charge, stability, and disulfide mutants. In vitro biophysical characterization of certain mutants will be performed, namely determination of thermal stability and chromatographic ion exchange retention equilibria. The foldase PDI and active site mutants of PDI will be over-expressed to determine the impact of individual foldase activities (i.e., redox vs. chaperone) on BPTI processing. Combining what has arguably been the most thoroughly studied model protein (in vitro) with the powerful genetic methodologies of a single-celled eucaryote should enable rapid acquisition of fundamental insights into the mechanisms of protein quality control in the secretary pathway.
