Collagen secretion is mediated by COPII vesicles. Recent work has suggested that the ubiquitination of one of the cage-forming subunits of the COPII coat, Sec31, may permit large vesicle formation to accommodate proteins such as procollagen. I am trying to reconstitute and optimize the sorting of procollagen into transport vesicles that bud from the ER in vitro to understand the mechanisms of large vesicle formation.
Peroxisomes are ubiquitous eukaryotic organelles which serve in beta-oxidation of fatty acids, hydrogen peroxide-based respiration and oxidative stress defense. In my study, an in vitro vesicle formation assay will be set up to identify ER-derived pre-peroxisomal vesicles. Furthermore the molecular mechanism will be studied by fractionating any cytosolic proteins (e.g. Pex19) required for budding of pre-peroxisomal vesicles.
Mammalian cells secrete proteins mainly through the conventional ER–Golgi secretory pathway. However, some cytoplasmic proteins are released in an unconventional manner independent of ER and Golgi. Autophagy, a cellular process characterized by de novo formation of double membrane structures for intracellular material degradation, contributes to one aspect of unconventional secretion, such as the secretion of IL-1beta. However, the molecular mechanism of autophagy-mediated protein secretion is unclear. Using IL-1beta as a marker, I plan to establish a cell-free system to uncover the mechanism of autophagy-related unconventional secretion.
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