You are studying the protein Hrd1, which has been implicated in rheumatoid arthr

Question

You are studying the protein Hrd1, which has been implicated in rheumatoid arthritis. Hrd1 is a multi- pass transmembrane protein that detects misfolded proteins as part of the “unfolded protein response” and tags them with Ubiquitin. One of the misfolded proteins that it can ubiquitinate is called MHC1. The functional Hrd1 protein has 6 transmembrane domains, but it lacks the traditional N-terminal ER signal sequence.

d) You want to determine whether these mutant Hrd1 proteins are, indeed, functionally defective. Based on what you know of its function, briefly describe a biochemical experiment to test if mutant Hrdl proteins are non-functional. (4 pts) e) In addition to promoting ubiquitination of misfolded proteins, Hrd1 can also act as a translocator to move ubiquitinated substrates back into the cytoplasm. Once there, where do ubiquitinated proteins usually go? What happens to them? (2 pts)

Explanation / Answer

d.Hrd1 is the core structural component of a large ER membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. the signal sequences direct the insertion of proteins into the membrane of the endoplasmic reticulum and are usually cleaved off by signal peptidase. Now if the protein in question Hrd1 lacks a signal sequence then it will not insert itself into the endoplasmic reticulum membrane. The proteins that are retained in the ER are retranslocated into the cytosol my protein importing machinery. In the cytosol, these are degraded by the proteasome, in most cases following polyubiquitination.

By understanding the function of ER sequence it is clear that if HRD1 protein lacks ER sequence then it will be unable to insert itself in ER and there will be no ubiquitination of MHC1 protein. As such misfolded protein MHC1 will remain in the cytosol and not be degraded. The principle of this experiment should emphasize on this fact that if there is no degradation of the target protein MHC1 and if it remains intact that means HRD 1 protein is non-functional. Obviously original protein and misfolded protein share lot of differences which could be a key feature in designing an experiment which could be the separation on the basis of charge by electrophoresis or any other criteria like reaction of protein that can produce different colors with the same product (i.e misfolded protein produce the different colored product than the original protein).

e. Ubiquitin acts as a marker for the proteolysis of targeted proteins.  Proteins are marked for degradation by the attachment of ubiquitin to the amino group of the side chain of a lysine residue. Such polyubiquitinated proteins are recognized and degraded by a large, multisubunit protease complex, called the proteasome. Such polyubiquitinated proteins are recognized and degraded by a large, multisubunit protease complex, called the proteasome. All these processes require energy in the form ATP. Thus actually the ubiquitin-marked proteins are degraded in the cytosol.

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