Specific ubiquitination of substrates requires the coordinated activity of three classes of enzymes commonly referred to as the E1 ubiquitin activating enzyme, the E2 ubiquitin
conjugating enzymes and the E3 ubiquitin ligases (Pickart, 2001) (Glickman and Ciechanover, 2002) (Hochstrasser, 1996) (Weissman, 1997) (Hershko and Ciechanover, 1998). The E1 catalyzes the activation of ubiquitin by hydrolyzing ATP to AMP and utilizes the energy to produce a high energy thioester bond between its active site cysteine residue and the NH3- terminal glycine of ubiquitin. The activated ubiquitin is transferred from the E1 to an active site cysteine residue in the conserved Ubc domain of the E2 Ubiquitin Conjugating enzyme. An E3 ubiquitin ligase functions to coordinate the transfer of the activated ubiquitin to a specific substrate by facilitating the transfer (Hershko and Ciechanover, 1998).
The E1 enzyme is highly conserved among eukaryotes and is represented by a single gene. In mammalian cells there are approximately thirty E2 enzymes that have been identified. The specificity of interactions between E2s and their cognate E3s is not fully understood. In many cases, the interactions are determined experimentally by conducting biochemical assays that lead to the identification of components in E2:E3 complexes. Several E2 have been generally categorized according to their association with specific systems, or according to their sub- cellular localization, usually relative to information obtained in yeast studies. For example, the Yeast Ubc4/Ubc5 Family of E2s is generally categorized as a family of E2s that function to degrade short-lived and abnormal proteins (Seufert and Jentsch, 1990) (Girod and Vierstra, 1993), and yeast Ubc6 and Ubc7 are categorized as membrane localized E2s based on their subcellular localization to the ER membrane (Biederer et al., 1997) (Lenk et al., 2002). There are hundreds of E3 enzymes in the mammalian cell suggesting the high level of specificity these enzymes confer to the ubiquitin system. There are two known classes of E3 Ubiquitin Ligase enzymes, HECT (Homologous to E6-AP Carboxyl Terminus) E3s (Huibregtse et al., 1995) (Cyr et al., 2002) (Pickart, 2001) and RING (Really Interesting New Gene) E3s (Cyr et
al., 2002) (Freemont, 2000). The HECT domain E3s have a HECT domain that functions similar to an E2 Ubc domain, having an active site cysteine that can be charged with an activated ubiquitin molecule that is delivered by an E2 (Cyr et al., 2002). The NH2-terminus of each HECT domain E3 is specific for a particular group of substrates while the COOH- terminus functions to recruit specific E2s and facilitate ubiquitin chain elongation. RING E3s are structurally and functionally distinct from HECT E3s (Cyr et al., 2002). They contain a RING domain that is formed and structurally stabilized by coordinated ionic interactions between zinc and histidine or cysteine residues that are properly spaced (Hatakeyama and Nakayama, 2003). The RING domain functions to transiently recruit E2s to the E3:substrate complex suggesting a scaffold-like function for the RING E3s. The mechanism of ubiquitin transfer to the substrate is not known. RING E3s that can directly interact with substrates using a substrate recognition domain are considered single-subunit RING E3s while those that require an adaptor protein or substrate selector that specifically recognizes the substrate are considered multi-subunit RING E3s (Cyr et al., 2002).
The RING domain family has recently been expanded to include a sub-class of U-box proteins that have a U-box domain in contrast to a RING domain (Cyr et al., 2002). The structural similarity between the RING domain and the U-box domain has been substantiated by computer based structural modeling techniques (Aravind and Koonin, 2000), and more recently by NMR structural data generated with the U-box domain of Prp19 interacting with a fragment of its cognate E2, UbcH7 (Ohi et al., 2003). The data indicate that not only is the structure of the RING and U-box domains conserved, residues important for interactions between RING domains and their cognate E2 correlate with residues in the U-box domain that are important for their interaction with cognate E2s (Ohi et al., 2003). Thus, the U-box domain
is predicted to function as a non-canonical RING domain, but the structure of the domain is stabilized by hydrogen-bonding and ionic-bridging interactions in contrast to coordinated zinc ionic interactions found in RING domains (Ohi et al., 2003). It has been hypothesized that U- box proteins function differently from RING E3s and perform as an E4 poly ubiquitin chain elongation factor. This hypothesis is supported by the inability of the U-box containing protein, yeast Ufd2, to independently facilitate ubiquitin chain assembly on a substrate. However, Ufd2 is required for efficient poly ubiquitination and degradation of artificial substrates when the target substrate is first ubiquitinated by the HECT E3 Ufd4 (Koegl et al., 1999). Other U-box E3s such as CHIP, CYC4, Prp19 and UIP5 in addition to CHN1, a C. elegans homologue of CHIP, have been shown to function with E2 ubiquitin conjugating enzymes, and independent of other E3s, to specifically target substrates for efficient poly ubiquitin chain assembly (Cyr et al., 2002) (Hatakeyama and Nakayama, 2003). Thus, U-box proteins represent a new sub-class of the RING type E3s (Hatakeyama and Nakayama, 2003). 1.5.2d Ubiquitin System and ER Membrane Proteins
Until 1993, the Ubiquitin System was thought to target soluble cytosolic and nuclear proteins for degradation. Around that same time, questions concerning the mechanism by which misfolded mutant ER proteins are selected for degradation were being addressed. These concerns arose from studies indicating that misfolded proteins and misassembled protein subunits that are retained in the ER are degraded in a manner that seemed instantaneous, having no apparent accumulation of degradation intermediates in the cytosol, and no association with the endosomal compartment. The first piece of evidence to address these concerns came from a study indicating a role for the Ubiquitin System in the degradation of a mutant ER protein. The study demonstrated that defects in protein translocation, caused by a temperature sensitive
mutation in Sec61 that promoted its degradation at increased temperature, could be suppressed by mutating Ubc6 (Sommer and Jentsch, 1993). Thus, a connection between degradation of abnormal proteins in the ER and the Ubiquitin System was identified. Since this discovery, much research has focused on the mechanisms by which a variety of ER membrane proteins are selected for degradation by the Ubiquitin System, and how that ubiquitinated substrate is degraded by the proteasome.