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.? ? Enteroviruses are members of the Picornaviridae family of positive strand non-enveloped RNA viruses, and include a number of important human pathogens such as poliovirus, coxsackievirus and echovirus. Despite the fact that enteroviruses (and other non-enveloped viruses) are not enclosed by a membrane, their replication never-the-less depends completely on host cell membranes, although the molecular details of this requirement are not understand. Enteroviruses are implicated in a wide range of human diseases including poliomyelitis, meningoencephalitis, encephalitis, pancreatitis and myocarditis. It is estimated that throughout the world, one billion human infections per year are caused by enteroviruses. All enteroviruses have a similar RNA genome of approximately 7.5 kb that encodes four capsid proteins and a number of nonstructural proteins. These non-capsid proteins are involved in viral replication and in virus-induced host cell membrane reorganization. One of the most dramatic effects of enterovirus infection is the massive reorganization of intracellular membrane systems, which ultimately involves all internal cell membrane systems except mitochondria and the nuclear envelope. The membrane structures produced upon viral infection are absolutely required for viral replication, which takes place on the membrane surface. Membrane reorganization occurs within two to three hours after viral infection. Subsequently, at three to four hours post-infection, enteroviruses completely inhibit intracellular protein transport. This effect can be produced by expression of only the viral 2B (or its precursor 2BC) protein in mammalian cells, or the viral 3A protein alone. The ability of the 2B(C) protein to inhibit transport is linked to its ability to rearrange membranes of the secretory pathway, but the 3A protein blocks transport by a mechanism that is not dependent on membrane reorganization. The transport inhibition and replication functions of the 3A protein can be genetically separated by mutations in 3A that affect one or the other function independently. The capacity of enteroviruses to block trafficking is important for viral infectivity, probably by suppressing both innate and adaptive immune responses and by suppressing the apoptotic pathway induced by viral infection. Recent studies have demonstrated a role for the Arf GTPases in poliovirus and coxsackievirus infection. Arf1, Arf3 and Arf5 (but not Arf6) are activated and recruited to membranes in an in vitro poliovirus replication assay. In addition, Arf1 is relocated from the Golgi apparatus to virus-induced membrane structures between two and three hours post-infection. A role for the Arf GEFs in viral infectivity was initially suggested by the fact that replication of poliovirus is completely inhibited by brefeldin A (BFA). In collaboration with the groups of Dr. Frank van Kuppeveld and Dr. Ellie Ehrenfeld, we have obtained direct proof that the BFA-sensitive Arf GEFs have important functions in at least two stages of viral infection, and have begun to elucidate their precise roles in these processes.
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