The Role of ALIX during Multivesicular Body Biogenesis

The rate at which cells grow and divide is controlled by signals from their environment. Amongst the most important of these is in the form of circulating small proteins called growth factors. These bind to receptors located on the cell's outer surface (or membrane), causing them to alter their pattern of interactions with molecules inside the cell that control cell growth. Growth factor receptors therefore act as essential bridges between the cell exterior and interior. In order to prevent responses continuing in an uncontrolled fashion, receptors must be removed from the cell surface shortly after growth factor binding and sent to a specialised cellular compartment called the lysosome, where they are degraded. Receptors first move from the cell surface to an internal, membrane-enclosed compartment called the endosome. Here the receptor is concentrated into regions of membrane that bud inwardly into the content space of the endosome, and are then directed to the lysosome. Precisely how inward budding occurs is not known. This proposal aims to dissect the molecular mechanisms underlying this process by using a range of cell biological and biochemical techniques.

Activated mitogenic receptors such as the epidermal growth factor receptor (EGFR) are endocytosed and sorted to intralumenal vesicles within the multivesicular body (MVB). This process is essential for the effective down-regulation of mitogenic signalling. EGFR is ubiquitinated and as a consequence recognised by a series of 'ESCRT' (Endosomal Sorting Complex Required for Transport) complexes, which sequester it away from recycling cargo such as transferrin receptor and sort it to the MVB. Several factors have been shown to bind ESCRT complexes. One of these is Alix, though functional evidence that it is important for MVB sorting is very limited. To address whether Alix is indeed involved, we generated a chimaera between the transferrin receptor and a peptide that binds Alix directly. This chimaera is sorted to the MVB lumen, dependent on its ability to bind Alix. Moreover, we have found that Alix is important for the down-regulation of EGFR. These findings provide a starting point for dissecting the mechanism of Alix action. We will first define where in the MVB sorting pathway Alix acts. For this, we will undertake detailed phenotypic analysis of the effect of Alix depletion on cargo sorting, defining the compartment in which MVB cargo is blocked using electron microscopy. The localisation of cargo and the formation of structures such as intralumenal vesicles will provide important information about the site at which MVB sorting is blocked. We will then investigate the molecular mechanism of Alix function. Alix interacts with several proteins and other effectors in cell extracts, but the importance of these interactions for MVB sorting is not known. We will use a simple functional read-out to test which interactions are critical for downstream effects of Alix on the MVB pathway. We will also test the hypothesis that a key role of Alix is to regulate the oligomerisation of late-acting ESCRT proteins, perhaps in conjunction with membrane lipid binding.