Investigation of the requirements for transitional endoplasmic reticulum (tER) site structure and function in Saccharomyces cerevisiae.

摘要

Eukaryotic cells contain distinct membrane-bound organelles that house sets of specialized metabolic reactions. The network of controlled communications between organelles is known as the secretory pathway. Transport of proteins and lipids between organelles is mediated by membrane-surrounded vesicles. Molecular mechanisms of vesicle trafficking are conserved from yeast to man. Protein coats facilitate selective membrane deformation into vesicles and enrich the budding vesicles with secretory cargo. Vesicles containing secretory cargo bud from the donor compartments and fuse with specific acceptor membranes. The budding yeast Saccaromyces cerevisiae has served as a valuable tool for the identification of protein and lipid participants in anterograde ER-Golgi transport mediated by the COPII protein complex. ER membranes posses regions that are devoid of ribosomes and specialized for vesicle budding, known as ER exit sites, or transitional ER (tER) sites. Ongoing COPII coat polymerization drives budding in these regions. Although tER site dynamics have been studied extensively in many organisms, the molecular requirements for coat polymerization at tER sites remain uncharacterized. The work presented in this dissertation validates S. cerevisiae as a fruitful model for the study of molecular requirements for ER exit site formation, maintenance and function. GFP fusions of the COPII proteins Sec13p and Sec23p were used as tER site markers in live cells. A screen was conducted to investigate tER site morphology in cells containing conditional mutations or deletions in genes required at various stages of ER-Golgi trafficking. It was found that specific proteins that participate in vesicle budding are required for tER site formation, while proteins involved in cargo selection or vesicle fusion are not directly required. A previously unappreciated role of secretory cargo in COPII coat localization was also observed. Investigation of the roles of ER lipids in COPII coat recruitment to tER sites showed that pecific phospholipids may participate in both vesicle budding and COPII coat recruitment. This work contributes to our understanding of the molecular mechanisms of COPII coat assembly at tER sites and COPII vesicle formation.