UC Berkeley

The coat protein complex II (COPII) mediates ER-to-Golgi transport in protein secretion. Genetic diseases affecting COPII function have demonstrated a requirement for COPII in secretion of bulky cargos, such as the 300-nm procollagen I (PC1), which is 5 times the diameter of an average COPII vesicle. Although large COPII vesicles were previously observed in cells overexpressing KLHL12, a substrate adaptor of the E3 ligase CUL3, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated, and the mechanism of cargo packaging and vesicle enlargement required further elucidation.

The research presented in this dissertation reports the existence of bona fide large COPII carriers of PC1 with evidence from multiple advanced microscopy techniques. By developing a cell-free COPII vesicle budding reaction, we demonstrated that the capture of PC1 into large COPII vesicles requires COPII proteins and the GTPase activity of the COPII subunit SAR1. This reaction was then used to show the co-packaging of PC1 with its cargo adaptor TANGO1 and the SAR1 nucleotide exchange factor (GEF) SEC12 into large COPII carriers. Coordinated cargo-sensing by TANGO1 and vesicle size regulation by SEC12 through its GEF activity was further shown to be important for PC1 secretion. In an independent effort to understand how the CUL3-KLHL12 complex regulates the size of COPII and PC1 secretion, we discovered that monoubiquitylation of SEC31A can inhibit its stimulatory effect on SAR1-GTP hydrolysis. Together, the study of two independent regulatory pathways revealed a converging mechanism on COPII size regulation by adjusting the local concentration of SAR1-GTP.