The Golgi complex is the central sorting station for nearly a third of all proteins in eukaryotic cells, but how cells regulate the complex flow of material through this organelle remains largely unknown. Protein and membrane traffic at the Golgi is controlled by Arf GTPases that function by recruiting effectors to make outgoing vesicles and tether incoming vesicles. The master regulators that activate Arf GTPase pathways are Arf-GEFs (guanine nucleotide exchange factors). In order to understand the molecular logic of Golgi trafficking, we must understand how the Golgi Arf-GEFs are regulated to make the molecular decision of where and when to activate their substrate Arf proteins. We have uncovered regulatory mechanisms that govern the function of the trans-Golgi network (TGN)-localized Arf-GEF, Sec7. We discovered that Sec7 can switch between autoinhibited and activated states, and is regulated by direct interactions with the activated forms of four different Golgi GTPases: Ypt31/32 (Rab11), Ypt1 (Rab1), Arl1, and Arf1. This collection of interactions represents a previously unappreciated level of crosstalk between these prominent Golgi GTPase pathways. Key questions remain regarding the biochemical basis for Sec7 regulation and the cell biology underpinning these interactions. Furthermore, we have determined that Gea1 and Gea2, the Arf-GEFs that localize to early Golgi compartments, are regulated through mechanisms that are distinct from those of Sec7. Our long-term goal is to determine how cells regulate trafficking at the Golgi complex. In order to both broaden and deepen our mechanistic understanding of the regulation of Arf activation at the Golgi, we propose the following Aims for this project: 1) Investigate the intra-molecular and inter-molecular interactions that regulate Sec7. We will use our established in vitro assays to characterize a newly identified functional link between distinct Sec7 regulatory domains. Using a new assay for measuring Sec7 activity in vivo, we will explore the possibilities that Sec7 integrates multiple GTPase signals and can sense and respond to changes in Golgi cargo load. We will perform an intragenic suppressor screen to identify new functional connections between Sec7 regulatory domains. Finally, we will seek additional structural information regarding the Sec7 C-terminal regulatory domains. 2) Determine the mechanisms regulating localization and activity of the early-Golgi Arf-GEFs Gea1/2. In order to understand how trafficking is regulated at the early- Golgi, we will perform a comprehensive investigation of the mechanisms regulating Gea1/2 membrane localization and activity. We will utilize chimeras and temperature sensitive-mutants to identify and characterize the regions of Gea1/2 regulating localization and activity. We will combine in vitro and in vivo approaches to characterize the roles of Gea1/2 binding partners. Finally, we will seek structural information to explain the basis for Gea1/2 regulation. We expect the outcome of these studies will be new and refined mechanistic models for regulation of trafficking at the Golgi complex.

Public Health Relevance

(relevance) Many human diseases arise from defects in the sub-cellular membrane and protein sorting machinery and its regulators. This project investigates the function and regulation of important regulators of sorting that are essential for cell viability.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098621-07
Application #
9415454
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Faupel-Badger, Jessica
Project Start
2012-02-01
Project End
2021-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
7
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Cornell University
Department
Miscellaneous
Type
Organized Research Units
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Gustafson, Margaret A; Fromme, J Christopher (2017) Regulation of Arf activation occurs via distinct mechanisms at early and late Golgi compartments. Mol Biol Cell 28:3660-3671
Richardson, Brian C; Halaby, Steve L; Gustafson, Margaret A et al. (2016) The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase. Elife 5:
Thomas, Laura L; Fromme, J Christopher (2016) GTPase cross talk regulates TRAPPII activation of Rab11 homologues during vesicle biogenesis. J Cell Biol 215:499-513
Paczkowski, Jon E; Fromme, J Christopher (2016) Analysis of Arf1 GTPase-Dependent Membrane Binding and Remodeling Using the Exomer Secretory Vesicle Cargo Adaptor. Methods Mol Biol 1496:41-53
Tang, Shaogeng; Henne, W Mike; Borbat, Peter P et al. (2015) Structural basis for activation, assembly and membrane binding of ESCRT-III Snf7 filaments. Elife 4:
Paczkowski, Jon E; Richardson, Brian C; Fromme, J Christopher (2015) Cargo adaptors: structures illuminate mechanisms regulating vesicle biogenesis. Trends Cell Biol 25:408-16
Richardson, Brian C; Fromme, J Christopher (2015) Biochemical methods for studying kinetic regulation of Arf1 activation by Sec7. Methods Cell Biol 130:101-26
Paczkowski, Jon E; Fromme, J Christopher (2014) Structural basis for membrane binding and remodeling by the exomer secretory vesicle cargo adaptor. Dev Cell 30:610-24
McDonold, Caitlin M; Fromme, J Christopher (2014) Four GTPases differentially regulate the Sec7 Arf-GEF to direct traffic at the trans-golgi network. Dev Cell 30:759-67
Richardson, Brian C; Fromme, J Christopher (2012) Autoregulation of Sec7 Arf-GEF activity and localization by positive feedback. Small GTPases 3:240-3

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