| The unfolded protein response (UPR) is an interorganellar signal transduction pathway that monitors endoplasmic reticulum (ER) homeostasis. Whole genome transcriptional analysis in budding yeast has revealed 381 genes regulated by the UPR. About half of those genes encode functions throughout the secretory pathway including protein folding and modification, protein quality control, lipid synthesis, protein trafficking, and vacuolar (lysosomal) proteases. Previous experiments have shown that the UPR is essential for ER stress tolerance but how the upregulation of individual genes contributes to stress tolerance is unknown. To answer this question, I devised an approach that uncouples the expression of individual target genes from UPR regulation.;The KAR2 gene was selected as the first target for analysis. KAR2 encodes Kar2p/BiP, a highly conserved protein belonging to the Hsp70 family of molecular chaperones. Kar2p is the first UPR target discovered and plays multiple functions in the ER including protein translocation, protein folding, ER associated degradation (ERAD) and karyogamy. These essential functions (protein translocation and folding) raise an interesting conundrum. Since half of UPR targets encode proteins that enter the secretory pathway, Kar2p is, in fact, an essential facilitator of the UPR at its last stage, the synthesis of target proteins. How then, is it possible to activate the UPR when a key facilitator is also a target gene that is immediately needed to deal with the misfolded products of stress (ERAD)?;To address this question and to study the contribution of individual target genes to the ER stress response, we engineered a strain that specifically uncouples Kar2p regulation from the UPR without compromising the activation of other target genes. Under stress, Kar2p upregulation was essential for cell viability. I showed that Kar2p upregulation was required to rid aberrant proteins through both the ERAD pathway and the ER-to-vacuolar overload pathway. Surprisingly, the essential functions of protein translocation and folding were unaffected when Kar2p levels became limiting under stress. This strategy allows Kar2p to temporarily dispense with its role in ridding misfolded proteins to facilitate the UPR until homeostasis is restored. The physiological importance of this triage mechanism was apparent because a temporary disruption of Kar2p essential functions was lethal. In contrast, cells could fully survive a temporary disruption of Kar2p's quality control functions.;On the other hand, if Kar2p was limiting for prolonged periods, cells would die. I demonstrated that under stress, the clearance of misfolded proteins is essential. Accordingly, in the strain defective in upregulating KAR2 expression by the UPR, cells eventually died because they could not efficiently degrade misfolded proteins in the ER, this resulted in the accumulation of misfolded proteins in the ER, which caused cellular toxicity. In addition, I also found that a potential export signal in misfolded proteins was formed despite the misfolding of proteins and function in an ER export event. |
