Regulation of Membrane Dynamics by Sec7 Domain Arf Nucle

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This unit studies the Sec7 domain guanine nucleotide exchange factors (GEFs) for the Arf family of small GTPases. We are interested in the roles of these proteins in membrane dynamics and protein trafficking. The Arfs and the Arf GEFs are important regulators of both organelle structure and protein transport throughout the cell. Eukaryotic cells are characterized by their internal membrane structure, which is essential for the correct spatial organization of the many biochemical reactions that take place within cells. The nucleus is separated from the surrounding cytoplasm by the nuclear envelope, a double membrane structure that is continuous with the endoplasmic reticulum (ER). Transmembrane domain proteins, proteins destined for secretion, and soluble resident proteins of organelles such as the lysosome/vacuole are translocated across the ER membrane into the lumen of this organelle. From here they are transported to their final destination via the Golgi apparatus. The ER spreads throughout cells, and gives rise to multiple Golgi elements that in mammalian cells are transported via microtubules to a region adjacent to the nucleus, thus forming the Golgi apparatus. A major function of the Golgi apparatus is the post-translational modification of proteins traveling through it, and sorting of these proteins to their correct destination in the cell. We are focusing our attention on a subfamily of Arf GEFs involved in transport through the Golgi apparatus, both in budding yeast and in mammalian cells. A central question in cell biology is how the elaborate and dynamic structures of membrane systems are maintained in the face of constant trafficking into and out of each organelle. In particular, the way organelle structure is generated and maintained, and how structure is correlated with the underlying molecular events of protein sorting and membrane remodeling are pressing questions. Evidence that the Arf GEFs play a key role in membrane dynamics and organelle structure came from studies of the cellular effects of brefeldin A (BFA), a drug that has profound effects on organelles of the secretory and endocytic pathways in a wide range of cell types. BFA causes the complete and rapid disassembly of the Golgi apparatus and its fusion with the ER, as well as fusion of the trans-Golgi network with endosomes. These experiments were the first to show the incredibly dynamic nature of the Golgi apparatus, whose elaborate structure would seem to suggest a more stable state. We demonstrated that three Arf GEFs in yeast are the major targets of the drug in the yeast secretory pathway. We also demonstrated the mechanism of action of this drug. BFA binds to a normally very short-lived reaction intermediate in the exchange reaction, an Arf-GDP-Sec7 domain complex, and forms an abortive quaternary complex that prevents the reaction from proceeding to completion. This unusual mechanism of action provides a paradigm for development of novel drugs. Instead of the usual search for drugs competing for a given substrate, screens could be designed for drugs that block reactions through stabilization of reaction intermediates. We are using a combination of techniques, including yeast genetics, molecular biology, imaging of yeast and mammalian cells and biochemistry, to elucidate the roles of the Arf GEFs in protein transport and organelle structure. An important step towards understanding the mechanisms of membrane trafficking will be to define the roles of the Arf GEFs at the molecular level, through identification of interacting partners, elucidation of membrane localization mechanisms and analysis of Arf GEF mutants in vivo. We have identified a number of interesting partners of the Arf GEFs in both budding yeast (Saccharomyces cerevisiae) and in mammalian cells, and are currently characterizing these novel partners and their roles in protein trafficking and membrane dynamics.