Stages of PI(3)P and ubiquitin-dependent MVB sorting: Endosomal proteins destined for delivery into the hydrolytic lumen of the vacuole/lysosome are actively sorted into vesicles that bud into the lumen of the endosome during the formation of an MVB. This is a highly selective process, as only a subset of endosomal proteins are destined for the lumen of the vacuole/lysosome. Covalent modification of endosomal proteins with ubiquitin appears to be the predominant signal for targeting into the MVB pathway. Function of the MVB pathway requires the activity of a large group of gene products, the class E Vacular Sorting Proteins (Vps).
Site-selection for the MVB sorting reaction is determined by interactions between the endosomally-enriched lipid species phosphatidylinositol-3-phosphate (PI(3)P) and the FYVE domain of Vps27/Hrs. Vps27/Hrs additionally contains a ubiquitin interacting motif responsible for recognizing the ubiquitin moiety on MVB cargoes.
In addition, Vps27/Hrs recruits the ESCRT-I complex, that also contains a ubiquitin binding domain. This higher order complex, containing both Vps27 and ESCRT-I, is thought to initiate the sorting of MVB cargoes into this pathway via the recognition of ubiquitinated endosomal proteins. The function of additional class E Vps proteins (including ESCRT-II and ESCRT-III) is required downstream of Vps27/ESCRT-I in order for MVB sorting to occur.
It is thought that these proteins serve to concentrate cargo during the formation of vesicles that will bud into the lumen of the endosome. They are also responsible for the recruitment of accessory factors, such as the de-ubiquitinating enzyme Doa4 that is responsible for removing ubiquitin prior to sorting of cargo into the MVB pathway, thereby sparing it from degradation. We have used the model substrate Carboxypeptidase S (CPS) to identify ubiquitin as a cis-acting MVB sorting signal, as well as the trans-acting machinery responsible for executing the sorting reaction. However, it should be noted that all findings are applicable to receptor downregulation in organisms as divergent as yeast and mammals.