Synchronized membrane protein trafficking in Saccharomyces cerevisiae

Abstract

The secretory trafficking pathway holds an indispensable role in regulating biological functions and its malfunction is associated with the onset of debilitating diseases. S. cerevisiae has long been utilized as a model organism since secretory pathways are highly conserved amongst eukaryotes. Owing to availability of rapid and precise genetic techniques, yeast has been preferred over human cells in numerous fields of fundamental biology. However, techniques for synchronizing protein trafficking are limited in this versatile model organism. Boncompain et al. (2012) established an elegant protein synchronization method named Retention Using Selective Hooks (RUSH) in the mammalian cell model. RUSH is a two-stage assay that utilizes streptavidin hooks to retain secretory reporter proteins tagged with streptavidin-binding peptide in the endoplasmic reticulum. The reporter proteins can be released using biotin, enabling synchronized transport. A preliminary version of yeast RUSH has recently been established by Choe Lab. However, its application is limited to luminal proteins; synchronizing trafficking of membrane proteins has remained challenging. In this study, I optimized the yeast RUSH system for membrane proteins by positioning streptavidin hooks at the cytosolic face of the ER membrane. Furthermore, the ratio between the streptavidin hook and SBP-tagged reporter proteins was fine-tuned to enhance the visualization of the synchronized membrane protein trafficking.