UC Berkeley

The coat protein complex II (COPII) achieves cargo sorting and vesicle formation at the endoplasmic reticulum (ER) and aids in protein trafficking by the early secretory pathway. The flexibility of the coat machinery to accommodate diverse size and shaped cargo in order to meet the demands of the cell is not well understood. An example of a large cargo protein is collagen, which is synthesized as 300nm precursor fibrils of procollagen in the endoplasmic reticulum (ER). The molecular mechanism by which large procollagen fibrils are accommodated in canonical COPII vesicles of about 70nm remains unclear. Genetic and biochemical evidence for the role of COPII in collagen trafficking has been established in several studies. Recent collaborative work has shown that mono-ubiquitylation of Sec31 by the ubiquitin ligase complex Cul3-Klhl12 leads to the formation of large COPII vesicles (200-500nm) that drive the secretion of collagen. Amongst the follow-up questions from this study were, whether these enlarged vesicles are bona fide transport carriers of procollagen and what might the three-dimensional ultrastructure be. This dissertation work aims to characterize the role of large COPII coated vesicles in intracellular procollagen-1 trafficking. Using a combination of in vivo imaging techniques and in vitro cell-free reconstitution assays, I present evidence that addresses this question.