Targeting and translocation mechanisms in the secretory pathway of the yeast Saccharomyces cerevisiae

Foreign protein production from the yeast Saccharomyces cerevisiae is accomplished through the secretory pathway of the cell, which deposits the final product into the extracellular media. A leader region at the beginning of the foreign protein interacts with intracellular machinery, directing the protein into the secretory pathway. The first step of the pathway is targeting to and translocation across the ER membrane. Because this step is often the rate-limiting one, a better understanding of the mechanisms involved will allow further optimization of foreign protein production.The most highly studied targeting mechanism is mediated by a ribonucleoprotein called the signal recognition particle (SRP), which targets the nascent protein to the ER membrane while it is still being translated, thus initiating a mechanism of cotranslational translocation. In S. cerevisiae, SRP is not an essential complex and may be depleted from the cell to study alternate pathways. Using this depletion system, I studied the cellular response to the loss of the SRP-dependent pathway and found that the cytoplasmic 70 kDa heat shock proteins (Hsp70's), specifically the SSA1 gene, are induced before other general cytoplasmic stress proteins and to a much higher extent than is seen in a standard heat shock response. Thus indicating that SSA1 may be involved in an alternate targeting pathway. The Ydj1 protein is also induced before the general stress proteins, but not as massively as the Hsp70's.In addition, I combined the SRP depletion system with a translocation assay that distinguished between cotranslational and posttranslational translocation. Using these two systems, I studied the translocation characteristics of a model foreign protein secretion system consisting of 4 different leaders directing the protein bovine pancreatic trypsin inhibitor (BPTI) for secretion. The results indicated that the leader regions affect the overall productivity of foreign protein secretion and use the cotranslational and posttranslational pathways to different extents. An overall translocation defect occurs initially after the loss of SRP, but is partially recovered by the chaperone induction seen. Even for the most efficient leader only 42% overall was translocated, indicating that there is potential to improve overall secretion by increasing the amount translocated.U of I OnlyETDs are only available to UIUC Users without author permissio