The Arf-GEFs Gea1 and Gea2 integrate signals to coordinate vesicle trafficking at the Golgi complex
Gustafson, Margaret Ann
At the Golgi complex, the biosynthetic sorting center of the cell, the Arf GTPases are responsible for coordinating vesicle formation. The Arf-GEFs activate Arf GTPases and are therefore the key molecular decision-makers for both anterograde and retrograde trafficking out of the Golgi. In Saccharomyces cerevisiae, three conserved Arf-GEFs function at the Golgi: Sec7, Gea1, and Gea2. Our group has described the regulation of Sec7, the trans-Golgi Arf-GEF, through autoinhibition, positive feedback, dimerization, and interactions with a suite of small GTPases. However, we lack a clear understanding of the regulation of the early Golgi Arf-GEFs Gea1 and Gea2. Here, I present insights into this regulation. I demonstrate that Gea1 and Gea2 prefer neutral over anionic membrane surfaces in vitro, consistent with their localization to the early Golgi, and that Gea1, Gea2, and Sec7 each localize to different Golgi compartments. I also show that a critical mass of either Gea1 or Gea2 is required for the essential role which Gea plays in vivo. Using in vitro membrane binding and catalytic activity assays, I show that the C-terminal domains of Gea1 and Gea2 toggle roles in the cytosol and at the membrane surface, preventing membrane binding by Gea in the absence of a recruiting interaction but promoting maximum catalytic activity once recruited. In vivo assays highlight the importance of these domains in recruitment of Gea to Golgi compartments and cell viability. I have identified the small GTPase Ypt1 as a recruiter for Gea1 and Gea2 in vitro, and I demonstrate that this interaction is dependent upon the C-terminus of Gea. I have also uncovered an additional interaction between Gea2 and the Golgi SNARE Gos1 which may provide the specific interaction required to target Gea1 and Gea2 to different compartments. I describe additional preliminary results and propose future experiments that will advance this line of research. This evidence evokes a model for intricate regulation of function and localization for Gea1 and Gea2 and raises interesting questions about the roles Gea1 and Gea2 play in Golgi trafficking. My findings not only illuminate core regulatory mechanisms unique to the early Golgi Arf-GEFs, but also pave the way for a clearer understanding of the coordination of Golgi membrane trafficking across transport events controlled by small GTPases, their regulators, and their effectors working in concert.
Cellular biology; Molecular biology; Biochemistry; Arf; Arf-GEF; Gea1; Gea2; Golgi complex; membrane trafficking
Fromme, Joseph Chris
Alani, Eric E.; Brown, William J.
Biochem, Molec & Cell Biology
PHD of Biochem, Molec & Cell Biology
Doctor of Philosophy
dissertation or thesis