Consider a cell surface receptor that upon ligand binding, becomes mono-ubiquiti

Question

Consider a cell surface receptor that upon ligand binding, becomes mono-ubiquitinated, undergoes endocytosis, and finally degradation via the multivesicular body pathway. You probe the system using siRNA to deplete cells of various components and observe the following experimental outcomes.

(1) Knocking down either the the relevant ubiquitin ligase or Hrs or tsg101 causes a lack of degradation of the receptor. Confusingly, the receptor continues to undergo rapid endocytosis upon stimulation, but steady-state surface levels of the receptor are only negligibly reduced.

(2) Knocking down various other components of ESCRT1, ESCRT2, or ESCRT3 results in permanent removal of receptor from the plasma membrane. However, it still does not get degraded. Instead it accumulates intracellularly.

(3) Knocking down vps4 causes a complex phenotype. The receptor undergoes endocytosis upon stimulation as nor- mal. Initially there is some receptor degradation indicated by a slight drop in the total cellular receptor level. However, after this the total receptor population is stable and oddly, as in part 1, at steady state almost 100% of the receptor population is found on the cell surface.

Explanation / Answer

FUNCTION OF : ubiquitin ligase is to attach a ubiquitin to the protein (here receptor); Hrs is to sort ubiquitinated proteins in early endosomes (this would decide the fate of the protein); tsg101 is to aid ESCRT cokplex to identify ubiquitinated proteins.

1) Therefore, knocking any of these down would interfere with the ability of the endosome to identify ubiquitinated proteins and sending them for degradation. Endocytosis is induced by binding of the ligand which is happens normally and so does recycling. As a result of which the number of surface receptors is maintained.

2) The ESCRT complexes are vital for the decision making and fate of the endosome. So, the receptor gets internalised but since the endosome is unable to decide as to whether to degrade or recycle it stays in the cytoplams reducing the number of receptors on the surface.

3) VPS is the protein which via its UEV doamin helps the ESCRT complexes to identify the ubiquitinated proteins. Its absence would cause the disability of the endosomes to select and set apart the ubiquitinated proteins from the non- ubiquitinated ones and hence all the internalised receptors would end up getting recycled as happened in the 1st case.

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