(134g) Analyzing the Stress Response Pathway in Saccharomyces Cerevisiae

Our research program has focused on identifying critical cellular interactions during the unfolded response during protein overexpression in the yeast Saccharomyces cerevisiae. Our laboratory seeks to use a systematic approach that goes beyond the empirical observations and optimizations common to more traditional studies, in order to establish a fundamental understanding of the key interactions in these systems.

We are studying overexpression of single-chain antibody in S. cerevisiae. Prior studies of many investigators show that the antibody is retained in the endoplasmic reticulum due to slow folding, and the antibody is bound by two cellular proteins, yeast BiP (Kar2) and PDI. The accumulation of unfolded protein activates the unfolded protein response and subsequently shuts down antibody expression. Using the known interactions with cellular proteins, we developed a mechanistic model that predicted that BiP overexpression would lower cell stress and improve antibody secretion. Although antibody expression was higher when BiP levels were increased experimentally, the stress response was only marginally ameliorated. Using chemical stressors, we also found that BiP overexpression had very modest effects on cell stress.

These data suggest that BiP has only a minor role in the stress response pathway activation, in contrast with earlier models of BiP-regulated dimerization of the Ire1p receptor, the ER stress sensor. Our data is consistent with recent studies of Kamara and colleagues, who have found that deletion of the BiP-binding site in Ire1p does not impact the stress response of cells.

Recently, we have investigated the effect of BiP interactions on scFv folding and secretion by making mutations to the scFv by site-directed mutagenesis, with the goal of identifying sites that might alter the interactions with BiP. Sites with identified either via a rational approach based on an algorithm for BiP binding, or using a random PCR mutagenesis approach combined with a surface display screen for enhanced scFv secretion.

We then compared the effects of BiP binding as determined by co-immune precipitation, ER stress, and protein folding. We found that in variants with decreased ER stress and improved secretion there is no change in BiP interaction. An increased BiP binding interaction with a variant correlated with decreased secretion, possibly due to BiP's role in the misfolding and ER degradation pathway. The implications of these results in the stress response pathway will be discussed.