Mitotic regulation of the Golgi apparatus- the role of the ARF nucleotide exchange factor GBF1

All cells from animals, plants and fungi are made up of different compartments, each with a unique composition and specific functions. Material is transported between these many of these compartments, or organelles, in membrane-bound packets called vesicles. This process, referred to as membrane traffic, is required for the correct functioning of cells, and organisms as a whole. For example hormones, antibodies, neurotransmitters, and the major components of skin, cartilage and bone are released from cells in vesicles that fuse with the cell surface, while growth factors and dead cells are removed from the bloodstream in vesicles that are taken up from the cell surface. One of the major compartments in cells is the Golgi apparatus, a collection of flattened membrane sacks called cisternae that are layered on top of each other to form stacks. The Golgi apparatus has two major functions: it is responsible for modifying sugar chains present on proteins and lipids; and the packaging of these molecules into transport vesicles for delivery to the cell surface or other compartments in the cell. Proper modification and delivery of proteins is of fundamental importance. Defects in Golgi function, and membrane traffic in general, are responsible for a number of human diseases. Furthermore, components of the membrane traffic machinery, including those at the Golgi apparatus, are hijacked by certain bacteria and viruses, allowing these pathogens to replicate and/or avoid detection by the immune system. It is therefore important we understand how the Golgi apparatus functions at the molecular level. When cells divide organelles are inherited by the daughter cells. For the Golgi apparatus, this involves fragmentation into smaller structures, and requires the modification of key Golgi components. We have recently identified one of these components as GBF1, a protein known to be important for membrane traffic at the Golgi apparatus. The modification of GBF1 during cell division suggests it plays an important role in Golgi inheritance. In this project we aim to investigate how GBF1 is modified, and how this modification is important for the division of the Golgi apparatus and cells as a whole. This will increase our knowledge of how cells divide, which is relevant to many diseases including cancer. We also plan to determine how GBF1 is recruited to the Golgi, which is important for our understanding of membrane traffic in all cells, including those that are not dividing. Finally, since GBF1 is targeted by viruses such as poliovirus, our work will provide new insights into how these viral pathogens influence GBF1 function, which will inform better design of therapeutic strategies against them.

The Golgi apparatus lies at the heart of the secretory pathway, receiving newly synthesised proteins and lipids from the ER, processing and then packaging these molecules into carriers for delivery to their subsequent destinations. When mammalian cells divide, trafficking is arrested and the Golgi is extensively fragmented, a process that is required for inheritance of this organelle by the daughter cells. Although the mechanisms are ill defined, it is known that transport arrest and Golgi fragmentation (and therefore inheritance) are driven by phosphorylation of Golgi targets. We have recently identified the nucleotide exchange factor GBF1 as a novel mitotic phosphoprotein. GBF1 acts upon ARF GTPases, promoting their membrane association and activation, and subsequent recruitment of effectors including the COPI vesicle coat required for trafficking at the Golgi apparatus. GBF1 is a target for enteroviruses that bind and affect its recruitment to membranes, resulting in altered Golgi membrane dynamics. The identity of the membrane receptor for GBF1 is unknown. We have found that GBF1 dissociates from Golgi membranes in mitosis, consistent with the loss of membrane bound ARF1 and COPI under these conditions. We hypothesise that GBF1 is a key target for mitotic kinases, and that its phosphorylation contributes significantly to membrane traffic arrest and Golgi fragmentation in mitosis. Furthermore, given the apparent role of ARF1 in other mitotic events, we believe that GBF1 phosphorylation will contribute more widely to mitosis. This project aims to investigate the regulation of GBF1 in mitosis and its significance for Golgi disassembly and mitotic progression. This will greatly increase our understanding of these fundamental processes. We also plan to use a number of combined strategies to identify the GBF1 receptor, a key component of the membrane traffic machinery and one that is highly relevant to the study of enteroviruses.