Determining the specificity of vesicle traffic at the Golgi apparatus

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The cells that make up our organs and tissues are comprised of internal compartments, called organelles, that have distinct compositions and functions. Most organelles contain fat-like molecules called lipids that make a limiting membrane to separate the organelle contents from the rest of the cell, as well as many types of proteins. The function of organelles requires the delivery of new materials and the exchange of materials with other organelles in the cell. Transport of proteins and lipids between organelles is mediated by small spherical carriers called vesicles, which bud off one compartment and bind to and fuse with their destination compartment to deliver their contents. This process, which is conserved in all plants and animals, is essential for life, and when defective can result in a large number of diseases in humans. It is also exploited by pathogenic bacteria and viruses during their life cycle. Vesicle transport is highly specific, such that vesicles are recognized at the destination compartment in a selective manner, which ensures they deliver their contents to the correct place. Although vesicle transport has been studied for decades, we still lack a good understanding of how vesicle recognition occurs.The Golgi apparatus is a major transport hub in the cell. It receives vesicles from other organelles, and also moves cargo between its own sub-compartments in vesicles. Its major function is to sort cargo for distribution, and to modify it so it matures correctly. Vesicle recognition at the Golgi is mediated by long proteins called golgins, which are act like tentacles to capture, or tether, vesicles at their ends. Previous work has shown that golgins act in a selective manner to tether vesicles, thereby contributing to the specificity of vesicle transport at the Golgi. However, what they recognise on vesicles is not known. It is also not known whether the golgins have overlapping specificity in vesicle recognition. This study will address these outstanding questions, focussing on the golgins that mediate transport within the Golgi, which is critical for the function of this organelle. Our preliminary data suggests that lipids on the vesicle surface dictate the specificity of vesicle transport at the Golgi through selective recognition by the golgins. To test this hypothesis, we will investigate the lipid binding specificity of the golgins, using purified lipid vesicles and golgins, combined with unbiased lipid identification techniques, and determine the features of the golgins that bind to lipids. Subsequently, we will determine the importance of golgin-vesicle lipid interaction in the transport and modification of cargo proteins at the Golgi apparatus. This will be achieved using gene editing techniques to alter the golgins so they can no longer bind vesicle lipids, and effects upon cargo transport assessed using established assays. Cargo modification will be also assessed using established methods to measure the amount and composition of sugars added to the cargo proteins in the Golgi, which is highly dependent on vesicle transport rates at this organelle. To assess the importance of golgin binding to vesicle lipids in a more physiological context, we will perform similar experiments in the nematode worm C. elegans. Effects upon development, viability, tissue formation and function, and ageing, will be assessed alongside analysis of the Golgi in different cell types. Because of the genetic tractability of this model, we will also be able to knock-out or modify the golgins in different combinations to assess the extent of overlapping specificity and functional redundancy between these proteins, and hence of vesicle transport at the Golgi. The work will be important for our understanding of vesicle transport, and specifically in how the specificity of vesicle transport is achieved, which represents a major unanswered question in the field.