Novel effectors of multivesicular body sorting

The behaviour of cells within a tissue is controlled by their environment. Amongst the most important signals that cells receive are from circulating small proteins called growth factors. These bind to specific proteins, called receptors, that are found on the surface of cells. Binding of growth factors causes the receptors to alter their pattern of interactions with many molecules inside the cell that control cell growth. In this way growth factor receptors act as essential bridges between the cell exterior and interior to stimulate so-called mitogenic responses, which enable cells to grow and divide. In order to prevent these responses continuing endlessly, which would lead to uncontrolled cell division, the growth factor receptor must be sent to an environment where it can no longer communicate with other cellular contents. Ultimately, it is sent to a specialised compartment within the cell, called the lysosome, where it is destroyed. Movement, or trafficking, of the receptor from the cell surface to the lysosome involves the receptor being sequestered into regions of the cell surface membrane that invaginate and pinch off to form spherical packages, or vesicles, within the cell interior. These vesicles first move to and coalesce with an intermediate compartment called the endosome, which is rather like a balloon. Importantly, the growth factor receptors are still active when they reach the endosome. To ensure they are stopped from working, they are enclosed within little vesicles that are forced within the inside of the endosome. This occurs by a process of inward budding, rather like poking deep impressions into a balloon and imagining these could pinch off to form internal packets. This process means that the mitogenic receptors are now completely separated away from the rest of the cell contents and unable to work. The endosome, along with these internal packages, is then sent to the lysosome. The aim of this project is to understand how activated mitogenic receptors, once they reach the endosome, are packaged into the interior of the compartment. The project will focus on identifying the function of a new protein involved in this process, and ask how delivery of new vesicles to the endosome is coupled to the formation of internal vesicles, so the whole process of receptor inactivation works with maximum efficiency.

Activated EGFR receptors are endocytosed and delivered in endocytic vesicles to the early endosome. EGFR is ubiquitinated and as a consequence recognised by a series of 'ESCRT' complexes, which sequester it within intralumenal vesicles inside the multivesicular body (MVB). This transit through the endocytic pathway is essential for the effective down-regulation of mitogenic signalling. We have identified two proteins, TSG101 and HDPTP, as essential components of ESCRT-dependent MVB sorting. We now show that a protein of unknown function, UBAP1, binds HDPTP and is a novel component of this pathway. Based on structural similarities between UBAP1 and the ESCRT-I component MVB12, we will test the hypothesis that UBAP1 binds TSG101, and indeed may represent a component of a specific subset of ESCRT-I complexes selectively involved in MVB sorting. This is important, since recent studies indicate that ESCRTs have divergent cellular functions, and only some are required for MVB sorting. Identifying such specific components is crucial for understanding mitogenic receptor downregulation. We will also test UBAP1 function at the endosome by careful phenotypic analysis of UBAP1 disruption, using strategies that are well established. One so-far unexplored topic is how the various stages of endocytic transport are coupled with each other, to ensure that receptor transit through the pathway is efficient and responsive to changes in flux. A further aim of this project is to examine one potential coupling mechanism. We have found that HDPTP binds the Rab5 effector and endocytic fusion regulator, Rabaptin-5, and influences its post-translational modification. We will test how TSG101, UBAP1 and HDPTP influence endocytic vesicle fusion by careful morphological examination of disruption of these proteins, focussing on the accumulation of endocytic vesicles. We will then examine how the binding of these proteins might influence Rabaptin-5 activity, including Rab5 GEF activity.

There are potential long term benefits to health in the long term from the knowledge that will be obtained through this research given the importance of EGFR downregulation on the impact of disease. Medical industry may be interested in ways to treat hereditary diseases associated with defects in this pathway, and in particular in the linkage of UBAP1 to human diseases. The primary means of informing this community of our work is through publication in the scientific literature. In addition, the PI and RA will all be expected to play an active role in disseminating information to increase the impact of the research. They will present their work at both national and international conferences. The PI and RA will also disseminate their work to a broader public. The function of the endocytic pathway is a topic that will be of general interest to the public, mainly because of the immediate visual impact of the work, and particularly the EM tomogram models. It is also a process that should engage with the public given the recent national debate about the availability of anti-EGFR family chemotherapeutic drugs such as Herceptin. Of particular relevance, Woodman has links with the Manchester Museum, who organise science days regularly, and several members of his laboratory have participated in Science Fairs in Manchester. One of the primary outcomes will be specialist skills training for the named RA, Ling Zhang. Ling has been trained in electron microscopy during the course of his PhD and this grant will provide further opportunities for specialist training using state-of-the-art instruments and under excellent guidance by Dr Alex Mironov.