Endosomal sorting: deciding the fate of surface membrane proteins (360G-Wellcome-212246_Z_18_Z)

Membrane proteins destined for lysosomal degradation are ubiquitinated within the endosome and then sorted into intralumenal vesicles (ILVs), to form the multivesicular body (MVB). This critically important process is exemplified by the sorting of EGF receptor (EGFR). MVB sorting requires ESCRTs (Endosomal Sorting Complexes Required for Transport). ESCRTs collectively recognise ubiquitinated EGFR on the cytoplasmic face of the endosome and capture it within ILVs, whilst they escape. Towards understanding how ESCRTs overcome this topological problem, we will reconstitute the process. We have identified all those ESCRTs that drive EGFR sorting, and how they bind each other. We will now reconstitute MVB sorting, using proteoliposomes containing EGFR and exploiting our full complement of baculovirus-expressed ESCRTs. We will use site-directed photo-crosslinking to map the entire process biochemically, and will complement this with further in vitro analysis of the molecular architecture within the developing ILV. Key conclusions will be verified in cells. Current ideas suggest ubiquitination is the determining factor for EGFR sorting. However, we believe instead that EGFR signalling-dependent activation of ESCRTs is decisive. We will systematically identify ESCRT post-translational modifications (PTMs) that map with MVB sorting, and test using both reconstituted proteoliposomes and in cells how these PTMs control the pathway.