Synchronized protein trafficking in Saccharomyces cerevisiae

• Main Author: Choo, Winifre Yun Xian
• Other Authors: Choe Young Jun
• Format: Final Year Project
• Language: English
• Published: Nanyang Technological University 2023
• Subjects: Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/172425
• Institution: Nanyang Technological University

In 2012, researchers developed an innovative technique employing streptavidin hooks to retain secretory reporter proteins tagged with streptavidin-binding peptide within endoplasmic reticulum (ER). This technique enables synchronized protein transport, as the proteins trapped in the ER can be released simultaneously by introducing biotin. Biotin disrupts the interaction between the streptavidin hook and binding protein. Saccharomyces cerevisiae (Baker’s yeast) is a well-established model organism for investigating fundamental biological processes in eukaryotes. While yeast has been extensively employed to investigate the secretory pathway, no method has been established in this organism to visualize the real-time protein trafficking under physiological conditions. Given the versatility of yeast as a model organism, introducing novel methods is expected to greatly advance research. Thus, in this project, I aimed to implement a novel protein trafficking visualization system in yeast. The first prototype of this system in yeast proved unsuccessful, displaying no co-localization between mCherry-tagged hook and eGFP-tagged reporter. Through a series of experiments and troubleshooting, adjustments were made to several parameters to optimize the protocol. As a result, successful retention and release of reporters from the ER lumen were observed, showcasing that this system was successfully optimized and implemented in yeast. The system implemented in this study would serve as a valuable tool to gain new insights into the secretory pathway and further our understanding of vital cellular processes.