The coat protein I (COPI) complex is important for membrane traffic and compartmental organization in the secretory pathway, but the biological roles of COPI are still remarkably uncertain. Even though COPI is postulated to be essential for secretion and for Golgi cisternal maturation, yeast COPI temperature- sensitive (ts) mutants show surprisingly mild defects: secretory traffic is blocked only for some cargo proteins, and cisternal maturation is slowed but not arrested. In other studies, displacement of COPI from membranes alters Golgi architecture, but the underlying mechanism is unknown. We propose to clarify the action of COPI in Saccharomyces cerevisiae. Our main approach is a refined version of the "anchor-away" method. Addition of rapamycin causes a tagged protein to be sequestered at an anchor site, thereby functionally inactivating the tagged protein. Preliminary data confirm that COPI can be sequestered and inactivated by rapamycin within minutes. In addition, we are optimizing a method for correlative fluorescence and electron microscopy of yeast. The working hypothesis is that (a) COPI is essential for intra-Golgi traffic because COPI vesicles drive cisternal maturation, and (b) COPI negatively regulates homotypic fusion at the early Golgi.
Specific Aim #1 is to test the role of COPI in Golgi cisternal maturation. The hypothesis is that COPI vesicles recycle resident Golgi proteins from older to younger cisternae, thereby driving cisternal maturation. By combining the anchor-away method with our established video microscopy technique for visualizing yeast Golgi dynamics, we will test whether cisternal maturation requires COPI. The prediction is that when COPI is inactivated, cisternal maturation will "freeze".
Specific Aim #2 is to test the role of COPI in traffic through the secretory pathwa. The hypothesis is that all protein traffic through the secretory pathway requires COPI. Secretory traffic will be assayed by pulse-chase analysis. The prediction is that inactivation of COPI will completely block secretory traffic by preventing the recycling of key trafficking components such as SNAREs and ER export receptors.
Specific Aim #3 is to test the role of COPI in Golgi homotypic fusion. The hypothesis is that in addition to promoting vesicle formation, COPI restrains the homotypic fusion of early Golgi membranes. In this context, COPI may negatively regulate Golgi cisternal size and Golgi fenestration. We will test these ideas using fluorescence and electron microscopy. The prediction is that a controlled reduction of Golgi-associated COPI will enhance homotypic fusion. For over a decade, the function of COPI has been one of the central mysteries in the secretion field. New yeast tools will enable us to make major inroads into this problem.

Public Health Relevance

Abnormal secretory pathway function is a causative agent in diseases such as cancer and developmental disorders. Adequate treatments will require a cell biological understanding of the organization and operation of secretory compartments. The proposed study aims to reveal these basic principles.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM104010-01A1
Application #
8577338
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Ainsztein, Alexandra M
Project Start
2013-09-15
Project End
2017-05-31
Budget Start
2013-09-15
Budget End
2014-05-31
Support Year
1
Fiscal Year
2013
Total Cost
$343,316
Indirect Cost
$122,921
Name
University of Chicago
Department
Genetics
Type
Schools of Medicine
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Papanikou, Effrosyni; Glick, Benjamin S (2014) Golgi compartmentation and identity. Curr Opin Cell Biol 29:74-81
Fitzgerald, Ivy; Glick, Benjamin S (2014) Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting. Microb Cell Fact 13:125
Glick, Benjamin S (2014) Integrated self-organization of transitional ER and early Golgi compartments. Bioessays 36:129-33
Bhave, Madhura; Papanikou, Effrosyni; Iyer, Prasanna et al. (2014) Golgi enlargement in Arf-depleted yeast cells is due to altered dynamics of cisternal maturation. J Cell Sci 127:250-7