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Protein Ubiquitination

Ubiquitin is a compound which is present inside all nucleated mammalian cells, and which can be chemically bound to specific programs. This binding modifies the properties of the tagged or “ubiquitinated” protein, directing it to specific parts of the cell and thereby controlling its ultimate fate. Ubiquitination is an essential process which is necessary for mammalian cells to behave normally, and a disruption in this process for an extended period of time will almost certainly be lethal.

Polyubiquitination

Polyubiquitination is required for the targeting of misfolded proteins to the proteasome for degradation (Sigismund et al., 2005). In order for misfolded proteins located in the lumen of the ER to undergo such targeting, they must exit the ER in a process known as retrotranslocation. While the process has not been fully elucidated, retrotransloaction relies upon a protein complex consisting of derlin-1, VIMP, the p97 ATPase complex, and Ufd1/Npl4 – a ubiquitin-recognizing protein cofactor (Ye, et al., 2004). Both a functional retrotranslocation complex and the polyubiquitination of target proteins are necessary for proper retrotranslocation to occur (Jarosch, et al., 2002). Consequently, misfolded proteins in cells lacking functional ubiquitin ligases will not be polyubiquitinated, and thus will not undergo retrotranslocation. These misfolded proteins will thus accumulate in the ER and cause ER stress, ultimately activating the unfolded protein response (UPR). The resultant phenotype is expected to mimic the phenotype noted in derlin-1 depleted C. elegans by Ye, et al., as both derlin-1 and polyubiquitination are necessary for proper retrotranslocation to occur.

Monoubiquitination

While polyubiquitination is generally associated with a single function, monoubiquitination is central to a number of related functions intracellularly. In yeast, for example, Katzmann et al. demonstrated that ubiquitination of K8 of the vacuolar hydrolase protein carboxypeptidase S (CPS) is necessary and sufficient for proper sorting of this protein into the multivesicular body (MVB) pathway, ultimately localizing to the lumen of the vacuole (Katzmann, et al., 2001). In addition to being required for proper vacuolar sorting, recent studies have demonstrated the requirement of ubiquitination for sorting of E-cadherin to lysosomes for degradation, suggesting a general mechanism of vacuolar/lysosomal protein sorting reliant upon ubiquitin modification of targeted proteins (Palacios, et al., 2005). Thus, a lack of functional ubiquitin ligases in cells would lead to significant defects in MVB/lysosomal sorting, resulting in mislocalization of numerous vacuolar/lysosomal proteins. This will likely result in the localization of these proteins to the membranes of their respective compartments (rather than proper lumenal localization), as was observed in the case of CPS-GFP lacking K8 (Katzmann, et al., 2001). Ultimately these defects would diminish lysosomal and vacuolar functioning due to a lack of properly-localized proteins, producing various deleterious effects on affected cells.

Monoubiquitination has also been shown to be a signal for clathrin-independent internalization in HeLa cells (Sigismund, et al., 2005). At low doses of epidermal growth factor (EGF), the EGF receptor (EGFR) is internalized via a clathrin-dependent mechanism and is not ubiquitinated whereas at high EGF doses EGFR is ubiquitinated and internalized via clathrin-independent mechanisms (Sigismund, et al., 2005). This ubiquitination and internalization is associated with a decrease in EGFR levels which may stem from the increased targeting of endocytosed receptors for degradation. It is likely that this serves as a mechanism by which the cell can prevent excessive signaling in response to large amounts of signal, conserving cellular resources and adapting to current physiological conditions with time. Additionally, ubiquitination of rescued ΔF508 CFTR protein leads to protein internalization and degradation, implicating a role for ubiquitination of membrane-associated proteins in the detection of misfolding in vivo (Sharma, et al., 2004). Consequently, cells lacking functional ubiquitin ligases would not internalize receptors via clathrin-independent mechanisms, as observed in the ubiquitination-deficient Y1045F cells used by Sigismund et al. This would likely lead to widespread defects in internalization of misfolded proteins and receptor molecules, particularly in response to high levels of extracellular signal, resulting in excessive signaling and impairing normal cellular functionality and adaptability.

Ubiquitination is also involved in a number of cellular processes not discussed in class, and as such it is unlikely that cells lacking any functional ubiquitin ligases would be viable. Nonetheless, if viable these cells would display the abovementioned defects in trafficking and would be significantly functionally impaired as a direct result thereof.

Sources

Cai Z, et al. Differential Sensitivity of the Cystic Fibrosis (CF)-associated Mutants G551D and G1349D to Potentiators of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl– Channel. J Biol Chem. 2006; 281: 1970-1977.

Cong M, et al. Binding of the β2 Adrenergic Receptor to N-Ethylmaleimide-sensitive Factor Regulates Receptor Recycling. J Biol Chem. 2001; 276: 45145-45152.

Jarosch E, et al. Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nature Cell Biology. 2002; 134-139.

Katzmann DJ, et al. Ubiquitin-Dependent Sorting into the Multivesicular Body Pathway Requires the Function of a Conserved Endosomal Protein Sorting Complex, ESCRT-I. Cell. 2001; 106: 145-155.

Palacios F, et al. Lysosomal Targeting of E-Cadherin: a Unique Mechanism for the Down-Regulation of Cell-Cell Adhesion during Epithelial to Mesenchymal Transitions. Molecular and Cell Biology. 2005; 25(1): 389-402.

Pedemonte N, et al. Small-molecule correctors of defective ΔF508-CFTR cellular processing identified by high-throughput screening. J Clinical Inves. 2005; 115(9): 2564-2571.

Proesmans M, Vermeulen F, Boeck KD. What’s new in cystic fibrosis? From treating symptoms to correction of the basic defect. Eur K Periatr. 2008; 167: 839-849.

Sharma M, et al. Misfolding diverts CFTR from recycling to degradation: quality control at early endosomes. J Cell Biol. 2004; 164(6): 923-933.

Sigismund S, et al. Clathrin-independent endocytosis of ubiquitinated cargos. PNAS. 2005; 102(8):2760-2765.

Wang X, et al. COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code. J Cell Biol. 2004; 167(1): 65-74.

Ye Y, et al. A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature. 2004; 429: 841-847.


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