Stress response mechanisms: Characaterization of gp96 oligomers and comaparative study on effects of stress on compatible solutes and heat shock protein levels in yeast

Glycoprotein 96 (gp96), an endoplasmic reticular chaperone, undergoes oligomerization when subjected to heat shock (above 42°C). ATP inhibits oligomer formation, and its hydrolysis is not required for this effect (McQuade and Thorne, 2004). Previously in our lab, two site-directed mutants D149A and E103A, in which single residues expected to be involved in ATP binding (D149A) or hydrolysis (E103A) were generated for the purpose of exploring the structure-function relationships in these phenomena. The basic aim of our study is to explore the structural origins of ATP's effect on gp96 oligomerization. We attempted to express the mutants in the GS115 strain of Pichia pastoris so that they could be characterized in in vitro studies; our attempts to do so were unsuccessful. Our study also aims to characterize the functional aspects of oligomers compared to dimers. Using gp96 purified from rat liver tissue, four distinct oligomeric species were isolated via native gel electrophoresis followed by electroelution. Our attempts to determine the chaperone activities of the isolated oligomers via aggregation assays using citrate synthase were also unsuccessful.;In a separate study, our objective was to characterize the stress response of P. pastoris by measuring compatible solute levels in the organism. P. pastoris, a methylotrophic yeast used as an expression system for the production of recombinant proteins, and to compare the results to those for Pichia angusta, a temperature resistant strain, and Kluyveromyces lactis, another commercially available protein expression strain. The comparison studies suggested that K. lactis responds to heat shock and hyperosmotic shock similarly to P. pastoris, whereas P. angusta responds differently. Trehalose and arabitol levels increase significantly when exposed to hyperosmotic stress or addition of citric acid, and decrease upon treatment with dithiothreitol (reductive stress). Expression of the stress response proteins Hsp70 and Hsp104 was up regulated by heat shock and hyperosmotic shock in P. pastoris and K. lactis, but not in P. angusta. The results of our work should aid in the optimization of yield and quality of recombinant proteins obtained using these expression systems.