In eukaryotes, vesicle formation at the Golgi complex is initiated by the conserved small GTPase Arf1. Arf1 is responsible for various trafficking pathways at the Golgi including retrograde recycling to the ER, intra-Golgi tracking, and exit from the Golgi at the TGN. These various pathways occurring at different stages of the Golgi require precious regulation of Arf1. In budding yeast, Arf1 localization and activity is controlled by three separate guanine nucleotide exchange factors (GEFs): Gea1, Gea2, and Sec7. The paralogues Gea1 and Gea2 are responsible for activating Arf1 at the early Golgi to commence COPI vesicle formation which is essential for retention of resident proteins at the endoplasmic reticulum and early Golgi. Yet, the mechanism by which Gea1 and Gea2 are recruited to the Golgi at the right place and time to activate COPI vesicle formation is unclear. Having a structure of the full-length protein would facilitate these efforts, however past attempts at solving the crystal structure have failed. In this poster I will present my work in determining the full-length structure of Gea2 and Gea2 bound to Arf1 using cryo-EM to 3.5A resolution. This data has enabled me to build atomic models of these proteins of which I have been able use to test various hypotheses of how these proteins are regulated and controlled.

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Muccini A, Fromme JC

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