Investigating Molecular Mechanisms of PRRC1 in Regulating COPII Uncoating and Collagen Packaging into COPII-Coated Carriers

Abstract

The eukaryotic secretory pathway plays important roles in protein secretion and in targeting various newly synthesized proteins to their specific resident compartments. The endoplasmic reticulum (ER) is the first station in the secretory pathway. Coat protein complex II (COPII) captures conventional cargo proteins into vesicles with approximately 70nm in diameter at the ER. Efficient ER export depends on the assembly and disassembly of COPII coats on ER membranes. Although significant progress has been achieved, cellular factors that regulate the assembly and disassembly of COPII still need to be further investigated. Moreover, large-size cargo protein procollagen, with its size over 300 nm and rigidity, poses a distinct challenge since it exceeds the conventional 70 nm diameter COPII vesicles.

By utilizing an in vitro vesicle formation assay in combination with quantitative MS analysis, my lab identified a cytosolic protein, PRRC1, present on the vesicle in a GTP-dependent manner. PRRC1 contains an N-terminal proline-rich domain and a C-terminal NTPase domain. The N-terminal domain mediates its interaction with COPII, while the NTPase domain inhibits the binding between SEC23A and Sar1A-GTP, thereby promoting COPII coat disassembly. Through this regulation of COPII dynamics, PRRC1 facilitates vesicle budding. In addition, I also identified cellular factors that are co-enriched with procollagen in transport carriers using the same methodology. Among these, an ER-located transmembrane protein VAPA regulates the disassembly of COPII inner coats by regulating ER cholesterol levels and knockdown of VAPA increases the collagen secretion. Another Golgi-located protein, Giantin is also involved in collagen carriers. Knockout of giantin inhibits the collagen secretion and ER-to-Golgi trafficking of RUSH-PC1 and endogenous PC1 by destabilizing COPII inner coats. I also identified the ER membrane mRNA-binding protein MTDH and the cytoplasmic chaperone CDC37, which may regulate PC1 translation and translocation into transport carriers. My results provide novel insights into the COPII uncoating and collagen trafficking process, also demonstrating the technique of vesicle formation assay is a robust and powerful tool to uncover novel factors involved in COPII-mediated export.

Date of Award	2025
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Yusong GUO (Supervisor)