Dissecting the roles of ZFPL1 and GMAP210 in Golgi biogenesis and membrane traffic

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 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. All membrane traffic steps involve a process called tethering, which is the initial attachment of the transport carrier to its destination compartment. There are many tethering factors in cells, including a family of related tethering proteins at the Golgi apparatus. Mutation of one of these proteins called GMAP210 has recently been shown to cause a lethal skeletal disease in humans. The mechanisms by which occurs are poorly understood, as are the details of how GMAP210 and other tethering factors work in healthy cells. The aim of this proposal is to determine how GMAP210 function is regulated, focussing on its association with another protein of the Golgi apparatus that we have recently identified. This work will inform us of how the fundamental process of tethering takes place inside cells, and how defects in this process can lead to skeletal and possibly other types of disease in humans.

The Golgi apparatus lies at the heart of the secretory pathway where it functions to modify, sort and package a huge variety of cargo molecules. The structure and function of the Golgi apparatus is dependent upon a number of proteins. Important amongst these are the golgins, a family of coiled-coil proteins that contribute to both the structural integrity of the organelle and the trafficking that occurs there. Golgins are thought to tether or link membranes together, and as such can be considered members of a wider family of membrane tethering proteins, which are important for most if not all membrane traffic steps that occur in the cell. One golgin that is of particular interest is GMAP210. GMAP210 is required for maintaining the Golgi ribbon, and for the efficient trafficking and processing of extracellular matrix components and proteins destined for cilia. The importance of GMAP210 has recently been highlighted with the discovery that mutations in gene encoding this protein lead to skeletal dysplasia. We have recently identified the zinc finger protein ZFPL1 as a GMAP210 binding protein, and our preliminary studies suggest ZFPL1 may act to regulate GMAP210-mediated tethering events. We propose to test this hypothesis using a number of in vitro and cell based approaches. Importantly we are in a position to dissect the mechanisms involved, and then to determine their importance to the structural organisation of the Golgi apparatus as well as the processing and trafficking of specific cargoes. This work will increase our understanding of how GMAP210 functions in cells, and more generally of how membrane tethering is regulated. The findings will also inform us of how defects in these processes can lead to human disease.

Wherever possible we will try to maximise the impact of our research. We will adopt several strategies to achieve this, as indicated below. Communications and engagement We will communicate our findings to the public via the open access University website and through the Faculty Research brochure. We will also notify the dedicated Faculty press officer of our findings at around the point of publication in academic journals. The press officer will then contact local and national news agencies, and if appropriate prepare press releases that these agencies can use. We envisage that findings of our current study will be of interest to the public at large given the clinical importance of GMAP210. My laboratory has participated in several public engagement initiatives and I plan for this continue. I have hosted two A level students in my group at different times as part of the Nuffield bursary scheme. The Faculty of Life Sciences continues to participate in the Nuffield scheme and I envisage hosting another A level student in my lab should the current application be successful. Members of my lab have participated in several public engagement exercises involving visiting schoolchildren which have been hosted at the Manchester Museum, with whom our Faculty has close links. Members of my lab will continue to participate in these exercises in the future. I have been involved closely with the Lowe Syndrome Trust, a registered UK Charity dedicated to supporting patients with the X-linked disorder Lowe syndrome and their families. While the current application is not relevant to Lowe syndrome, it is relevant to skeletal dysplasia. I will engage with colleagues in our Faculty (Mike Briggs, Karl Kadler, Ray Boot-Handford) that have connections with relevant support groups to explore possible public engagement with these organizations. Collaboration This application fits the BBSRC Strategic priority area of increased international collaboration. We have been in close contact with Dr Bruno Antonny at the CNRS in Nice, and we have agreed to initiate a collaboration to investigate the functions of ZFPL1 using a specialized in vitro assay. We envisage the collaborative part of the proposal lasting 8 months, which will be spent in France. We foresee the collaboration continuing beyond the current project. The collaboration will form the basis of a longer term investment of my group into synthetic biology, which means in this case reconstituting Golgi membrane trafficking and biogenesis events using purified components. The collaboration will therefore have enormous long term benefit to my group, as well as hopefully benefitting the work ongoing in the Antonny lab. Exploitation and Application The work outlined in the proposal is basic research. Thus it is not trivial to realize the short and long term benefit of the work in terms of direct commercial or clinical exploitation. However, the scientific data obtained will increase the knowledge of UK and international scientists, in the academic, clinical and commercial sectors. Given the relevance of the work to human disease, the findings will be of interest to those investigators and businesses looking to exploit such information for the development of therapeutics. The technical and intellectual knowledge acquired by the post-doctoral research assistant during the course of the proposed work will equip this person to pursue a career in science or science-related discipline and in this way contribute to the UK knowledge and skill base and ultimately the UK economy.