Characterizing Membrane Traffic in the Early Secretory Pathway Using the RUSH Retention System

Abstract

The early secretory pathway encompasses the endoplasmic reticulum (ER) and the ER–Golgi intermediate compartment (ERGIC) organelles. The ERGIC is now understood to be a complex cargo sorting hub involved in a variety of cellular and tissue processes, however the traffic pathways to and from the ERGIC are still unclear. Classical methods employed for the analysis of a cargo’s journey along the secretory pathway rely on reversible traffic blocks leading to cargo accumulation in the ER. Although these methods were key to characterize Golgi and post-Golgi traffic routes, their poor specificity to the cargo of interest and limited spatiotemporal resolution make them inadequate for the fine characterization of cargo traffic in the early secretory pathway. In this chapter, we describe a protocol to study the traffic of cargo proteins in the early secretory pathway using the Retention Using Selective Hook (RUSH) system, a highly specific and sensitive tracking system with a high spatiotemporal resolution. Taking GLUT4 and GLUT1 as examples of unconventionally and conventionally secreted cargo respectively, we describe the steps to clone the cargoes in the RUSH vector and follow and quantify their traffic along the early secretory pathway. This RUSH method can also be used to study the traffic of other cargo proteins in the early secretory pathway.