Biochemistry, Nottingham University
|Courseware Index| |Year
3| |Ubiquitin Index| |Ubiquitin Pathway|
3.0 Enzymes of the Ubiquitin Pathway
3.1 Ubiquitin-activating enzyme, E1
The first step in ubiquitinylation involves activation of ubiquitin, followed by transfer to a ubiquitin conjugating enzyme. This first step is carried out by the activating enzyme, or E1. Several genes for E1 from different species have now been cloned and sequenced and code for proteins of 115-125kD. Wheat, and possibly other organisms, contain genes for more that one E1 . The role of these different E1s is not understood. E1s contain a conserved cysteine at the active site and a nuleotide binding site. E1 is found in the nucleus and cytosol and associated with the cytoskeleton . E1 is also phosphorylated in mammalian cells by the kinase cdc2 , which presumably reflects the role of the ubiquitin in cell cycle control. E1 is also found to form complexes with ubiquitin conjugating enzymes and E3s .
3.2 Ubiquitin-conjugating enzymes
While there may be two or even three species of E1 in cells S. cerevisiae is currently known to contain at least 10 genes coding for ubiquitin-conjugating enzymes (E2). Why are there so many E2s? Because some at least of the specificity of ubiquitinylation appears to be dependent on E2s. New E2 enzymes from a growing variety of species are being discovered all the time. All contain a cysteine in a conserved domain of about 16kD (the UBC domain) which contains the UBC motif. This cysteine accepts ubiquitin from E1 to form a thiol ester. Substitution of this cysteine abolishes E2 activity . A suggested motif rich in basic residues is found at the N-terminus of the UBC domain which is may be involved in E1 binding .
E2s (UBC) can be classified on the basis of their structure into three classes.
- Class I: these proteins comprise simply the "UBC" domain. In vitro these enzymes are very poor at transferring ubiquitin to proteins on their own, and probably require an E3 to aid this in vivo. UBC 4 and 5 of S. cerevisiae and UBC1 or Arabidopsis thaliana are examples of this class of E2, and are known to important in the ubiquitination of many short-lived and abnormal proteins prior to degradation.
- Class II: these enzymes contain C-terminal extensions of the UBC domain. The extensions are different in type but very acidic extensions, as found in UBC2 (also known as RAD6) of S. cerevisiae appear to mediate inteaction with protein substrates, in this case with the basic histones. UBC2/RAD6 will ubiquitinate histones in vitro, which requires the C-terminal extension  and is known to be involved in DNA repair. This may be a form of ubiquitination that results in protein modification but not degradation. Other C-terminal extensions appear to be involved in E2 localisation. UBC6 of S. cerevisiae is found anchored to the ER membrane with the active site facing the cytosol. The 95 residue C-terminal extension of UBC6 includes a hydrophobic signal-anchor sequence.
- Class III: N-terminal extensions are present in this class of E2s. Several enzymes of this class have been identified but the function of the extensions is unknown .
At least one E2s does not fit into any of the three classes. A large (230kDa) E2 in reticulocytes (the precursor cells of erythrocytes) is expressed during erythroid differentiation and may be involved in remodelling events leading to the mature cell.
A multiple alignment of three UBCs, 2 Class I (UBC1 of A. thaliana and UBC4 of yeast) and a Class II (UBC2 of yeast) highlights the conserved regions and shows the C-terminal extension of UBC2/RAD6. The 3D crystal structures of UBC1 of A. thaliana and UBC4 of yeast have been solved and can also be viewed here.
3.3 Ubiquitin ligases - "recognins" or E3s
Some E2s appear to require no other protein factor, at least in vitro, to transfer ubiquitin to a suitable target. Others require an additional protein factor, an E3. The E3 binds the target protein, and presumably therefore selects the target. This implies E3 recognize a motif in the substrate protein that targets it for ubiquitinylation. It has been suggested that such motifs be called "degrons", degrons being any motif that targets proteins for degradation (by the ubiquitin-dependent or and other systems). An E3 can therefore be called a "recognin". Unfortunately these useful terms have not caught on and the term "ubiquitin-ligase" is still used for E3s. Until recently the proximal donor of ubiquitin to target proteins was thought to a ubiquitin charged E2 (UBC), and E3s were not thought to be directly involved in the transfer of ubiquitin to the substrate protein. This is indeed the case for one class of E3s. However, recently E3s have been discovered which form thiol esters with ubiquitin and these charged E3s are the proximal donors to targets. These E3s may also act as "recognins". To date then two classes of E3s have been identified:
- E3s which do not form thiol esters with ubiquitin. The recognition of some targets for ubiquitinylation relies upon the identity of the N-terminal residue of the target protein (see Section 5). There appears to be several E3s in reticulocytes responsible for "N-end rule" recognition, selecting substrates on the basis of the N-terminal residue. In S. cerevisiae a single gene for the N-end rule E3  has been identified and sequenced and shows no similarity to any other known protein. Deleting this gene has little effect on the mutant cells, which are capable of ubiquitin-dependent degradation of short-lived proteins. The significance of the N-end rule, therefore, is unclear. These E3s do not form thiol esters and must act as recognins, forming complexes with charged E2s and target proteins to facilitate ubiquitinylation. The E3s responsible for the N-end rule appear to act with UBC2/Rad6 type E2s . Ciechanover's E3. E6-AP.
- E3s which do form thiol esters with ubiquitin. The tumour supressor p53 is targeted for ubiquitin-mediated degradation following association with the human papilloma virus (HPV16) protein E6. E6 recruits a cellular protein E6-AP to promote ubiquitinylation . E6-AP was the first member of a family of novel E3s discovered which form thiol esters with ubiquitin and act as the proximal donor in target ubiquitinylation , in this case the target is p53 (see Section 5 for more details). Perhaps the nomenclature E3 is confusing here as the recognin in this case may be seen as the viral E6 protein which does not participate in ubiquitin transfer, and is more analagous in function to the E3 described above, rather than E6-AP. Similar proteins to E6-AP have been found in rat and yeast. The yeast enzyme, RS5p/Npi1p, is involved in ubiquitinylation of the yeast uracil permease, a cell surface protein (see Section 5). RS5p contains interesting features, such as a C2 domain, which may be involved in membrane association, and a putative protein binding domain (WW). These features can be viewed here.
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