Many human diseases arise from defects in sub-cellular membrane and protein sorting machinery and its regulators. The Golgi complex is the primary sorting organelle in eukaryotic cells, and proteins of the Arf GTPase family control virtually all traffic out of the Golgi complex. Arf GTPases are activated at the trans- Golgi network by the Arf-GEF (Guanine nucleotide Exchange Factor) proteins BIG1/ARFGEF1 and BIG2/ARFGEF2 in humans, and by the homologous Sec7p protein in yeast. Although the basic mechanism of Arf activation - nucleotide exchange by the GEF domain - is well characterized, how Arf-GEF function is regulated at Golgi membranes is not known. Our lab has recently discovered that Sec7p exhibits two distinct autoregulatory behaviors: autoinhibition and positive feedback activation;these behaviors depend upon regions of the N-terminus and C-terminus of Sec7p, including a novel membrane recruitment domain. The high degree of sequence conservation between the yeast Sec7p protein and its human homologs indicates that these regulatory behaviors are very likely to be relevant in human cells. The long-term goal of this project is to understand how the essential function of the Golgi-localized Arf-GEFs is regulated, and will be obtained by pursuing the following four Specific Aims: 1) Characterize the Sec7p autoinhibitory mechanism and the role of the N-terminus. We will use in vitro assays and mutagenesis to dissect the molecular mechanisms of autoinhibition and N-terminal function. 2) Characterize the molecular mechanism of positive feedback. In vitro assays will be used to characterize the interaction between Arf1p-GTP and the membrane recruitment domain of Sec7p, and to compare the affinity of this interaction to the affinity of Arf1p-GTP for its effectors. 3) Determine the role of Sec7p autoregulation in protein and membrane trafficking in vivo. Sec7p autoregulatory mutants will be tested for their in vivo effects on cell growth, Golgi morphology, Sec7p localization, and cargo sorting, using electron and fluorescence microscopy, classical biochemical trafficking assays, and live-cell imaging. 4) Test the generality of Golgi Arf-GEF autoregulation. We will use in vitro assays to determine if BIG1, BIG2, and GBF1 (the human early-Golgi-localized Arf-GEF) also exhibit autoregulatory behavior. The proposed research is critical to our understanding of membrane and protein traffic at the Golgi, as Arf-GEFs are master regulators of transport-carrier formation at this organelle. We expect that our research will lead to a clear model for how activation of Arf GTPases at the Golgi is regulated spatiotemporally.

Public Health Relevance

Many human diseases arise from defects in the sub-cellular membrane and protein sorting machinery and its regulators. This project investigates the function of an important regulator of sorting that is essential for cell viability.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM098621-01A1
Application #
8245998
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2012-02-01
Project End
2017-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
1
Fiscal Year
2012
Total Cost
$294,256
Indirect Cost
$99,256
Name
Cornell University
Department
None
Type
Organized Research Units
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
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
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
Richardson, Brian C; McDonold, Caitlin M; Fromme, J Christopher (2012) The Sec7 Arf-GEF is recruited to the trans-Golgi network by positive feedback. Dev Cell 22:799-810