The kinetics of protein folding and disulfide bond formation in the yeast endoplasmic reticulum will be studied, with the aim of attaining a quantitative understanding of those components of the lumenal environment which most directly influence the rate and efficiency of folding in vivo. A deeper understanding of folding in the ER will enable rational engineering of efficient heterologous production systems for pharmaceutical proteins. Misfolding in the secretory pathway is also an aspect of several human genetic and viral diseases, providing further motivation for these studies. The experimental system consists of Bovine Pancreatic Trypsin Inhibitor (BPTI) expressed in the yeast Saccharomyces cerevisiae. The depth of structural, kinetic, and thermodynamic knowledge available concerning BPTI folding in vitro makes this an attractive model protein, and the genetic malleability of yeast makes it a useful model eucaryote. Genetic analysis of yeast secretory mutants has yielded considerable insights into other aspects of secretion, and this system will provide a direct phenotypic probe of the folding environment in the ER, enabling detailed studies of cellular factors involved in folding. The central hypothesis is that the rate and pathway of BPTI folding in vivo differs qualitatively from the pathways which have been studied in detail in vitro, due to the presence of chaperones, foldases, membrane translocation, a regulated redox and ionic environment, and the presence of a pro region. The folding pathway of BPTI is affected in vivo by the availability of alternative intermolecular disulfide partners (e.g., protein disulfide isomerase), and chaperones which may reduce steric barriers to formation of buried disulfide bonds by stabilizing unfolded conformations. The rate and efficiency of BPTI folding in the yeast ER will be precisely measured using pulse-chase radiolabeling, immunoprecipitation, and trypsin-Sepharose affinity purification, and the distribution of folding intermediates will be determined electrophoretically. The role of lumenal calcium, ATP, pH, and regulated redox in BPTI folding will be explored by biochemically disrupting the cell's homeostasis for each of these variables. Pairwise alanine-for- cysteine replacements will be constructed to eliminate each of the three disulfide bonds in BPTI, and the consequences for folding determined in vivo. The role of both a yeast pro region and the native BPTI pro region in determining the rate and efficiency of BPTI folding in vivo will be studied. It has been determined that when BPTI is overexpressed in yeast, it accumulates intracellularly in an unfolded conformation. Particular chaperones (BiP, EUG1) and foldases (PDI, cyclophilin) will be overexpressed in an attempt to overcome this observed bottleneck in folding and secretion.
