The endomembrane system is the defining feature of eukaryotic cells and ensures that the appropriate proteins are sorted and transported to their correct subcellular location where they perform their biological functions. The coat protein complex II (COPII) mediates the formation of COPII vesicles, which are responsible for exporting proteins and lipids from the endoplasmic reticulum (ER). The core COPII coat consists of five proteins: the small GTPase Sar1, the Sec23/24 heterodimer, and the Sec13/31 heterotetramer. The recognition and subsequent packaging of cargo into COPII vesicles is mediated by interaction with the Sec24 subunit.
The development of a cell-free vesicle budding reaction has facilitated a biochemical analysis of COPII trafficking. Initial experiments examining COPII packaging of mammalian cargo proteins suggested that while some cargo proteins could be packaged directly by the core COPII components, other cargo proteins seemed to require additional cytosolic factors for efficient packaging. Whether auxiliary cytosolic factors exist to promote the COPII packaging of a specific subset of cargo proteins is the central question that motivated the work described in this dissertation.
This question was first approached using Vangl2, a cell surface localized transmembrane protein involved in planar cell polarity signaling, as a model cargo molecule. However, during the course of characterizing COPII packaging of Vangl2 in vitro, it was determined that this was not the appropriate system to address this question. As a result, proTGFα, the precursor form of transforming growth factor alpha, was chosen as a model cargo protein to continue to address the central question.
Here, I describe the key observations that show the existence of auxiliary cytosolic factor(s) that are required for efficient COPII packaging of specific cargo. In the particular case of proTGFα, both its recruitment by the COPII machinery and its final packaging into COPII vesicles require the cooperation of one or more auxiliary cytosolic factors and a transmembrane cargo receptor, Cornichon-1. Moreover, I present evidence showing that interaction of cargo proteins with the COPII coat is insufficient for packaging into COPII vesicles. This suggests that cargo recruitment and vesicle packaging are two distinct steps of COPII vesicle formation. Taken together, these observations provide novel insights into the molecular mechanism of COPII vesicle formation in mammals.