ERAD

Endoplasmic Reticulum Associated Protein Degradation (ERAD) designates a cellular pathway which targets misfolded proteins of the endoplasmic reticulum for ubiquitination and subsequent degradation by a protein-degrading complex, called the proteasome.

Mechanism

The process of ERAD can be divided into three steps:

Recognition of misfolded proteins in the endoplasmic reticulum

The recognition of misfolded proteins depends on the detection of substructures within proteins such as hydrophobic regions, unpaired cysteine residues and immature glycans.

In mammalian cells for example, there exists a mechanism called glycan processing. In this mechanism, the lectin-type chaperones calnexin/calreticulin (CNX/CRT) provide immature glycoproteins the opportunity to reach their native conformation. They can do this by way of reglucosylating these glycoproteins by an enzyme called UDP-glucose-glycoprotein glucosyltransferase. Terminally misfolded proteins, however, must be extracted from CNX/CRT. This is carried out by EDEM and ER mannosidase I. This mannosidase removes one mannose residue from the glycoprotein and the latter is recognized by EDEM. Eventually EDEM will target the misfolded glycoproteins for degradation.

Retro-translocation into the cytosol

Because the ubiquitin-proteasome system (UPS) is located at the cytoplasm, terminally misfolded proteins have to be transported from the endoplasmic reticulum back into cytoplasm. It seems that a protein complex, called Sec61, constitutes the channel necessary for the transport of these misfolded proteins. Further, this translocation requires a driving force that determines the direction of transport. Since polyubiquitination is essential for the export of substrates, it is likely that this driving force is provided by ubiquitin-binding factors. One of these ubiquitin-binding factors is the Cdc48p-Npl4p-Ufd1p complex.

Ubiquitin-dependent degradation by the proteasome

The ubiquitination of terminally misfolded proteins is caused by a cascade of enzymatic reactions. The first of these reactions takes place when the ubiquitin-activating enzyme E1 hydrolyses ATP and forms a high-energy thioester linkage between a cysteine residue in its active site and the C-terminus of ubiquitin. The resulting activated ubiquitin is then passed to E2, which is a ubiquitin-conjugating enzyme. Another group of enzymes, more specifically ubiquitin protein ligases called E3, bind to the misfolded protein. Next they align the protein and E2, thus facilitating the attachment of ubiquitin to lysine residues of the misfolded protein. Following successive addition of ubiquitin molecules to lysine residues of the previously attached ubiquitin, a polyubiquitin chain is formed. A polyubiquitinated protein is produced and this is recognized by specific subunits in the 19S capping complexes of the 26S proteasome. Hereafter, the polypeptide chain is fed into the central chamber of the 20S core region that contains the proteolytically active sites. Ubiquitin is cleaved before terminal digestion by deubiquitinating enzymes. This third step is very closely associated with the second one, since ubiquitination takes place during the translocation event. However, the proteasomal degradation takes place in the cytoplasm.

ERAD ubiquitination machinery

The ER membrane anchored RING finger containing ubiquitin ligases Hrd1 and Doa10 are the major mediators of substrate ubiquitination during ERAD. The tail anchored membrane protein Ubc6 as well as Ubc1 and the Cue1 dependent membrane bound Ubc7 are the ubiquitin conjugating enzymes involved in ERAD.

Checkpoints

As the variation of ERAD-substrates is enormously, several variations of the ERAD mechanism have been proposed. Indeed, it was confirmed that soluble, membrane and transmembrane proteins were recognized by different mechanisms. This led to the idenftification of 3 different pathways that constitute in fact 3 checkpoints.

*The first checkpoint is called ERAD-C and monitors the folding state of the cytosolic domains of membrane proteins. If defaults are detected in the cytosolic domains, this checkpoint will remove the misfolded protein.

*When the cytosolic domains are found to be correctly folded, the membrane protein will pass to a second checkpoint where the luminal domains are monitored. This second checkpoint is called the ERAD-L pathway. Not only membrane proteins surviving the first checkpoint are controlled for their luminal domains, also soluble proteins are inspected by this pathway as they are entirely luminal and thus bypass the first checkpoint. If a lesion in the luminal domains is detected, the involved protein is processed for ERAD using a set of factors including the vesicular trafficking machinery that transports misfolded proteins from the endoplasmic reticulum to the Golgi apparatus.

*Also a third checkpoint has been described that relies on the inspection of transmembrane domains of proteins. It is called the ERAD-M pathway but it is not very clear in which order it has to be placed with regard to the two previously described pathways.

Diseases associated with ERAD-malfunctioning

As ERAD is a central element of the secretory pathway, disorders in its activity can cause a range of human diseases. These disorders can be classified into two groups.

The first group is the result of mutations in ERAD components, which subsequently lose their function. By losing their function, these components are not longer able to stabilize aberrant proteins so that the latter accumulate and damage the cell. A very important example of a disease caused by this first group of disorders is Parkinson’s disease. It is caused by a mutation in the parkin gene. Parkin is a protein that functions in complex with CHIP as a ubiquitin ligase and overcomes the accumulation and aggregation of misfolded proteins.

In contrast to this first group of disorders, the second group is caused by premature degradation of secretory or membrane proteins. In this way, these proteins aren’t able to be deployed to distal compartments, as is the case in cystic fibrosis.

ERAD and HIV

As described before, the addition of polyubiquitin chains to ERAD substrates is crucial for their export. HIV uses an efficient mechanism to dislocate a single-membrane-spanning host protein, CD4, from the ER and submits it to ERAD. CD4 is normally a stable protein and is not likely to be a target for ERAD. However, HIV produces the membrane protein Vpu that binds to CD4. As Vpu is phosphorylated, it mimics substrates for the E3 complex SCF^βTrCP. In cells that are infected with HIV, SCF^βTrCP interacts with Vpu and ubiquitinates CD4, which is subsequently degraded by the proteasome. Vpu itself escapes from the degradation.

Questions

The big questions for ERAD are:

*How are misfolded proteins more specifically recognized?

*What is the channel for the retrotranslocation of luminal ER proteins?

See also

* endoplasmic reticulum
* ubiquitination
* proteasome
* protein folding
* oxidative folding

References

*Meusser B, Hirsch C, Jarosch E, Sommer T. Nat Cell Biol. 2005 Aug;7(8):766-72 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16056268&query_hl=1&itool=pubmed_DocSum ERAD: the long road to destruction]
*Ding WX, Yin XM. Sorting, recognition and activation of the misfolded protein degradation pathways through macroautophagy and the proteasome. Autophagy. 2007 Oct 19;4(2) PMID 17986870
*Shilpa Vashist and Davis T.W. Ng.(2004). [http://www.jcb.org/cgi/reprint/165/1/41 Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control.] The Journal of Cell Biology 165, 41-52
*Lloyd W. Ruddock and Maurizio Molinari.(2006). [http://jcs.biologists.org/cgi/reprint/119/21/4373 "N"-glycan processing in ER quality control.] Journal of Cell Science 119, 4373-4380