Determining the specificity of vesicle traffic at the Golgi apparatus

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.

The secretory pathway is fundamentally important, producing around 30% of all cellular proteins, including the vast majority of membrane and secreted proteins. The Golgi apparatus lies at the heart of this pathway, acting as a trafficking hub and major site of cargo modification. As with all trafficking steps in the cell, Golgi transport vesicles must be specifically recognized at their target membrane prior to undergoing fusion and content delivery. Vesicle recognition at the Golgi is mediated by the golgin family of tethering proteins, which extend into the cytoplasm to capture vesicles in a selective manner. The mechanism by which golgins recognize transport vesicles is currently unknown, and the extent to which the golgins have overlapping specificity in vesicle recognition is also unclear. Our preliminary data suggest that the golgins that mediate intra-Golgi traffic bind to vesicle lipids to mediate tethering. We therefore hypothesise that vesicle lipids confer specificity to intra-Golgi traffic. The project will test this hypothesis, as well as testing the extent to which the golgins share overlapping specificity in vesicle recognition. In vitro binding experiments and lipidomics will be used to determine the lipid binding preferences of the golgins and reveal the features of the golgins required for lipid binding. Cell-based functional experiments will then be carried out to determine the importance of golgin-lipid interaction in protein trafficking and glycosylation at the Golgi apparatus, using established assays. The in vivo importance of golgin-lipid binding will be assessed in the model organism C. elegans, which will also allow a systematic analysis of the physiological importance of golgin-mediated tethering as well as revealing the extent to which the golgins share overlapping functionality in vivo. Together, the results will reveal how transport vesicles are specifically recognized at the Golgi to mediate protein trafficking at this compartment.