Illustration 1. (above)
Ubroles: Ubiquitin has been shown to play a role in the targeting protein cargo during a number of intracellular protein sorting events. At the Golgi, the general amino acid permease (Gap1, indicated in orange) can either be targeted to the cell surface for function, or directly into the MVB pathway. Subsequent to functioning at the cell surface, Gap1 is ubiquitinated to drive its internalization into the endocytic pathway and subsequent degradation via the MVB pathway. Ubiquitinated endosomal proteins are targeted for inclusion into intralumenal vesicles during the MVB sorting reaction (indicated by orange Gap1 and green EGFR). Entry into the MVB pathway will ultimately result in delivery to the hydrolytic lumen of the vacuole/lysosome.
The life cycle of certain viruses also requires the MVB sorting machinery. The Gag protein of HIV-1 (indicated as blue circles) is required for productive replication of the virus. Ubiquitination of Gag has been shown to correlate with its sorting into nascent viral particles. Furthermore, the Gag protein recruits the MVB sorting machinery to drive the budding of viral particles from the host cell.
Illustration 2. (above)
Receptor Downregulation: The surface of a typical cell contains a large number of receptors that perceive extracellular signals and transduce the appropriate intracellular response. In the case of growth factor receptors, such as the epidermal growth factor receptor (EGFR), agonist binding induces a signaling cascade that stimulates growth. Concomitantly, activated receptors are targeted for degradation via the MVB pathway.
Growth homeostasis is modulated in part through a balance between signaling and downregulation. Defects in the function of the MVB pathway stabilize activated growth factor receptors, thereby prolonging their ability to signal to stimulate growth. This de-regulation of growth control has negative implications in the context of tumor growth and developmental patterning during organismal development.
Illustration 3. (above)
The MVB sorting pathway: During the formation of an MVB, cargoes destined for delivery to the lumen of the vacuole/lysosome are actively sorted into vesicles that invaginate and bud into the lumen of the endosome (giving rise to the characteristic “multivesicular” morphology). Heterotypic fusion of the MVB with the vacuol/lysosome results in the delivery of these intralumenal vesicles and cargo proteins contained therein to the hydrolytic lumen for degradation. Proteins that remain in the limiting membrane of the MVB will be delivered to the limiting membrane of the vacuole/lysosome following heterotypic fusion.
Illustration 4. (above)
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.
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