Investigation of the molecular mechanisms of Erv26p-dependent protein sorting.

摘要

Studies of the secretory pathway have revealed a multitude of secretory proteins that depend on the coat protein complex II (COPII) for export from the endoplasmic reticulum (ER) in transport vesicles. Some secretory proteins require protein sorting adaptors to provide an indirect link to the COPII coat. Protein sorting adaptors in ER-to-Golgi transport are generally thought to contain sorting signals that mediate binding to COPII as well as the retrograde-directed coatomer protein (COPI) coat for bi-directional trafficking between the ER and Golgi. However, mechanisms of protein sorting adaptor-dependent ER export remain unclear, including what fraction of secretory proteins require sorting adaptors, how many types of sorting adaptors are needed to accommodate the diversity of cargo, and the regulation of sorting adaptor-dependent transport. In this thesis, studies were performed on the uncharacterized S. cerevisiae ER vesicle protein known as Erv26p, and indicate that it is a novel protein sorting adaptor. Experimental evidence demonstrates that Erv26p is packaged efficiently into COPII vesicles, and strains lacking Erv26p show an ER accumulation of vacuolar pro-alkaline phosphatase (pro-ALP). pro-ALP interacts with Erv26p and is packaged into COPII vesicles in an Erv26p-dependent manner. To investigate the molecular mechanism of Erv26p-dependent sorting, mutations in conserved regions of Erv26p were made and analyzed. Erv26p interactions with both coat and cargo are required for efficient pro-ALP transport, and are independent of one another. Additionally, Erv26p homodimerization is important for pro-ALP interaction. The C-terminus of Erv26p is required for efficient COPII-dependent ER export, whereas the third luminal loop domain is involved in cargo recognition. These results have revealed mechanisms governing Erv26p-dependent transport, and future studies on this sorting adaptor-cargo pair should allow an elucidation of general mechanisms underlying receptor-mediated ER export.