Modulation of glycosylation homeostasis by

Project: Research project (funded)Research

Project Details


Vesicular sorting in the Golgi generates the characteristic differential localization of enzymes in the various Golgi cisternae. A critical role for vesicle targeting falls to tethering, since it is believed to be the first contact between vesicle and target membrane. This proposal aims at establishing details of the vesicle tethering process, and to provide some explanation how vesicle targeting influences enzyme localization, and thereby glycan chain synthesis. The intra-Golgi transport of vesicles that carry resident glycosylation enzymes is mediated, amongst others, by the COG tethering complex and Rab GTPases. We have found that multiple Golgi Rabs interact with COG, and hypothesise that these interactions will be important for the targeting of different vesicles in the Golgi. To test this, we will characterise the interactions between the Rabs involved in early Golgi targeting and COG. Through in vitro and in vivo interaction studies we will attempt to show how COG differentiates between the various GTPases to allow selective vesicle targeting. Moreover, by mutagenesis aimed at disrupting the COG-Rab interactions, we will generate tools for the dissection of intra-Golgi vesicle transport. Subsequently, we will specify the requirements for vesicle tethering at the early Golgi to initiate the molecular analysis of individual vesicle targeting reactions. We will group the COG interacting Rabs and golgins, a protein family involved in vesicle tethering at the Golgi, into separate protein sets required for individual tethering reactions. Finally, we will directly test the relationship between vesicle targeting mediated by individual COG-Rab pairs, and glycan synthesis. Using the mutants disrupting COG-Rab interactions we will interrupt specific targeting reactions, and analyse the resulting glycan defects. Through this we hope to establish a model explaining how the specific targeting of different enzyme-sets to Golgi cisternae can diversify glycan sequences.

Layman's description

Animal cells contain multiple membranous organelles, such as the nucleus, mitochondria, or the various organelles of the secretory pathway. The secretory organelles are responsible for releasing all the proteins from the cell that perform important external functions, such as communication, defence or the physical connectivity between cells. Appropriate sorting of these proteins between organelles is important to ensure that each reaches their correct destination, be it outside the cell, or in one of the organelles themselves to help the organelle's functions. Protein sorting is achieved by relatively small membranous structures, called vesicles, which pinch off one organelle to carry a selected set of its proteins to a second organelle, which is the preferred destination for those proteins. When a vesicle arrives at the target organelle, the two membranes have to be brought into close apposition, or tethered to each other. Subsequently the vesicle and target membrane will merge with each other to deliver the vesicle's cargo. We will study the process of vesicle tethering at the main sorting hub of the secretory pathway, the Golgi apparatus. The tethering of an important vesicle subset at the Golgi is assisted by a protein complex called COG, which is controlled by GTPases of the Rab family. There
are 70 different Rabs known in people, and all are known to shift their shape when hydrolysing a bound GTP molecule. This shape change allows them to control the function of other proteins associated with them, such as COG. We already know, that COG communicates with seven different Rabs.

An important unanswered question in vesicle tethering is, how the vesicles find the correct target organelle? We hypothesise that COG functions as a machine to power the tethering of a vesicle, while each Rab may steer COG to use the correct vesicle and tether it to the right target membrane, to allow faithful sorting of the vesicular cargo. The experiments proposed in this application will test this hypothesis by looking at the interplay of COG with three Rabs that we have previously shown to physically contact COG. The physical contact puts the Rabs into the correct position to control COG, but the exact nature of each contact, especially the differences between the various COG-Rab pairs will be important to explain how specific targeting occurs.

Vesicle tethering needs other factors besides COG and Rabs as well, and therefore a second question we will address is how those other factors, members of the golgin protein family, will fit into the picture. For the one machine, COG, there are about a handful Rabs, and there could be as many as a dozen golgins, which could also play their part in targeting specificity. As part of this study we will catalogue some of these golgins and Rabs into protein subsets that will form the basis for later more detailed investigations about their specific roles in vesicle tethering and vesicle targeting specificity.

The proteins delivered to the outside of the cell by the secretory pathway are often modified in various ways to enhance their functionality, most prominently by sugar chains built of nine different sugar building blocks. Most of the sugar chains, are generated by numerous enzymes that reside in specific sub-compartments of the Golgi, called cisternae. Sugar chains often require a specific sequence of the building blocks added to one another by a specific sequence of the enzymes. This in turn requires the enzymes to be sorted into the correct order. The function of the vesicles using COG is to sort these enzymes into specific cisternae to maintain a given order. This is very important, since defects in COG that cause defects in sugar chains, have been found to cause human diseases. As a final quest of this proposal we will focus our attention on how the different COG-Rab pairs mediate the sorting of enzymes to allow correct sugar chains to be built.

Key findings

Just as buses take people in a crowded city to their destination, proteins are guided to the different compartments of a cell via carriers called vesicles. As for buses, it is important that vesicles find their correct destination. Guidance for vesicles is largely determined while they approach their target, a process called tethering. We have investigated the proteins that guide vesicles through tethering at one specific cellular compartment, the Golgi Apparatus. These fall into three groups called COGs, golgins and Rabs. We found a network of combinatorial interaction between Golgi Apparatus associated members of these groups. This network is the ground stone for establishing which sets of these proteins participate in guiding vesicles to different regions of the Golgi Apparatus.
To follow on from this work we then wanted to understand how one subset of these interlinked proteins does its job to guide the vesicle. We found that COGs, which form one stable entity all together, bind at two ends of the golgin called TMF. TMF, as all golgins is very long, and can act like a fishing pole to grab the approaching vesicle. The COGs can then use three Rabs, Rab1, Rab2 and Rab6, which all bind both COGs and TMF, to reel in the vesicle along TMF by using first its linkage to TMF far from the Golgi, and then the linkage close by. Presumably TMF and Rabs specify where the vesicle goes, whereas COGs act as the machinery to move the vesicle along to the target.
Some progress was also made along the path to determine what the effect would be to perturb only one subset of the vesicle guiding clues. We hypothesised that such a perturbation would cause a small imbalance in the function of the Golgi, which is to assemble sugar chains in an orderly fashion, and thereby generate slightly altered sugar chains. We have developed a method to select altered form of one COG, COG4, which still links with the other COGs and some of the Rabs, but fails to link with one Rab, Rab30. Such an altered COG, if placed into cells will perturb the vesicle guidance step involving Rab30, but none of the others. The first generation of altered COG4 proteins were not yet useful because there was some knock-on effect on their linkage to other COGs and Rabs, but we have improved the selection procedure, and will now go ahead to produce the necessary COG4 versions. The perturbations in cells generated by the altered COG4 could be very informative in the future for studying disease states in which sugar chains are altered such as certain cancers and inflammatory conditions, and will help tissue engineering efforts in which the sugar chains are often of great importance.
Effective start/end date1/03/0822/04/11