Ubiquitin-mediated pathways in C. elegans^*

Ubiquitin (Ub) is a ubiquitously expressed and highly conserved 76 amino acid polypeptide (Hershko and Ciechanover, 1998; Figure 1A). The covalent tandem attachment of multiple Ub to a target protein to form poly-ubiquitin chains can mark the protein for degradation by the 26S proteasome. Ub is covalently attached to substrate proteins by the concerted actions of three classes of enzymes (Hershko and Ciechanover, 1998). A ubiquitin-activating enzyme (E1) uses one ATP molecule to bind Ub via a thiolester linkage. The activated Ub is transferred to a ubiquitin-conjugating enzyme (E2), also via a thiolester linkage. The E2 is brought to the substrate by binding a ubiquitin-protein ligase (E3) that binds both the E2 and the substrate. Once bound to an E3, the E2 either directly transfers Ub to the substrate or transfers the Ub through a thiolester linkage to the E3, which then transfers the Ub to the substrate. Multiple rounds of E2 interactions with substrate-bound E3 are required to produce a poly-Ub chain on the substrate. In a few cases, the E2-E3 combination is not capable of adding more than a few Ub, and in this situation a ubiquitin chain assembly factor (E4) is required for the conjugation of additional Ub to form a poly-Ub chain (Koegl et al., 1999).

Figure 1. A) Alignment of individual ubiquitin polypeptides from C. elegans (C.e.), H. sapiens (H.s.), and S. cerevisiae (S.c.). Differences between Ub residues are boxed. Note that C. elegans and humans only differ at one position relative to each other and at three positions relative to budding yeast. The locations of the seven lysines in Ub are marked with the residue numbers provided above the alignment. B) Translation of the C. elegans ubq-1 polyubiquitin gene. The amino acid sequence is presented at 76 amino acids per line so that individual ubiquitin repeats are aligned. Note that repeat 6 has a different amino acid at position 9 of the repeat (yellow box, isoleucine rather than threonine). There are two amino acids after the final ubiquitin repeat.

Ub is conjugated to target proteins or other Ub through a bond between the conserved C-terminus of Ub and the ε-amino group of a lysine residue on the target protein or other Ub (Hershko and Ciechanover, 1998). A minimum of four tandemly-attached Ub are required to allow recognition of the target protein by the 26S proteasome, presumably because a tetramer of poly-Ub assumes a higher order structure that is required for recognition (Pickart, 2000). Ub has seven lysine (Lys) residues (Figure 1A) and Ub can be conjugated to several of these Lys residues. Poly-Ub chains created by conjugation through a Lys-48 linkage targets a substrate for degradation by the 26S proteasome. In contrast, poly-Ub formed by Lys-63 conjugation does not lead to proteasome-mediated degradation, but instead is associated with the regulation of endocytosis or changes in target protein function (Schnell and Hicke, 2003). Similarly, conjugation of a single Ub (mono-ubiquitination) or less than four Ub can affect protein activity, including aspects of transcriptional regulation, protein trafficking, and endocytosis (Schnell and Hicke, 2003). The functions of poly-Ub chains created with Lys-11 and Lys-29 linkages have not been determined (Aguilar and Wendland, 2003).

Ubiquitin-mediated proteolysis is the most important pathway for the degradation of nuclear and cytosolic proteins. Inactivation of the Ub proteolytic pathway inhibits the degradation of the majority of cellular proteins, regardless of whether the proteins have short or long half-lives (Rock et al., 1994). Given the central importance of ubiquitin-mediated protein degradation in a range of cellular processes, it is not surprising that Ub-mediated pathways are important for multiple aspects of C. elegans development and cellular physiology.

The 26S proteasome is a conserved chambered protease complex that is present in both the cytoplasm and the nucleus (Wojcik and DeMartino, 2003). It consists of a 20S proteasome, a central core containing proteolytic subunits, and two 19S regulatory complexes that bind to ubiquitinated substrates, cleave off ubiquitin, and then unfold and translocate the substrate into the 20S core (Pickart and Cohen, 2004). C. elegans has 14 conserved subunits that comprise the 20S core, as well as 18 conserved 19S components (Davy et al., 2001). RNAi depletion of proteasome components during larval stages produces larval arrest and lethality while RNAi depletion in adult hermaphrodites produces progeny that arrest at the one-cell stage with defective meiosis I, indicating the central importance of this pathway (Takahashi et al., 2002; Gonczy et al., 2000).

There are two loci for ubiquitin (Ub) in C. elegans, ubq-1 and ubq-2. ubq-1 is a polyubiquitin locus (Graham et al., 1989). The predominant splice form of ubq-1 encodes an 838 amino acid peptide that includes 11 tandem Ub sequences (Figure 1B). The polyubiquitin structure of the locus is common to other eukaryotic species (Schlesinger and Bond, 1987). The polyubiquitin protein is post-translationally cleaved into individual Ub peptides by ubiquitin C-terminal hydrolases (Johnston et al., 1999). The Ub peptides of ubq-1 are identical with the exception of repeat 6, which substitutes an isoleucine for a highly conserved threonine at position 9 of the repeat (Figure 1B). The functional significance of this altered Ub peptide is not known. In C. briggsae, this atypical Ub repeat is not present, instead the orthologous polyubiquitin locus comprises ten Ub repeats that are all identical to the predominant C. elegans Ub sequence.

The second Ub locus, ubq-2, includes an intact canonical Ub fused to the L40 ribosomal large subunit protein (Jones and Candido, 1993). This fusion gene is broadly conserved in eukaryotes (Schlesinger and Bond, 1987). The ubq-2 locus contains the only copy of the L40 ribosomal subunit in the C. elegans genome. In yeast, the hybrid protein is rapidly cleaved to form Ub and the L40 ribosomal subunit (Finley et al., 1989). The transient presence of Ub in the fusion protein promotes the incorporation of the L40 subunit into ribosomes (Finley et al., 1989). Once cleaved, the Ub is functional for covalent attachment to proteins (Ozkaynak et al., 1987).

RNAi of ubq-1 or ubq-2 produces a one-cell stage arrest during the meiotic divisions, similar to inactivation of the proteasome (Gonczy et al., 2000; Piano et al., 2000). The relative importance of ubq-1 vs ubq-2 is not known, as RNAi is expected to inactivate both genes due to their extensive homology (Tijsterman et al., 2002).

As in other eukaryotes, there is only a single ubiquitin-activating enzyme in C. elegans, UBA-1. Disruption of UBA-1 activity would be expected to completely inactivate the Ub proteolytic pathway. However, while RNAi of uba-1 produces an embryonic arrest, it is not as penetrant as RNAi for ubq-1 or particular proteasome components, perhaps because, as an enzyme, it is more resistant to effects of depletion (Maeda et al., 2001; Kamath et al., 2003; Simmer et al., 2003; Piano et al., 2000; Gonczy et al., 2000).

There are 22 proteins with homology to ubiquitin-conjugating enzymes (UBCs) in C. elegans, with an additional three ubiquitin E2 variants (UEVs) that lack the critical cysteine residue in the catalytic site (Jones et al., 2002). The C. elegans UBCs are numbered ubc-1-3, 6-9, and 12-26; with numbers 4, 5, 10, and 11 skipped. The numbering of UBCs in C. elegans does not match that of S. cerevisiae or humans, and orthologous groupings are presented in Table 1. Note that ubc-9 and ubc-12 designate conjugating enzymes for the Ub-like proteins SUMO (SMO-1) and Nedd8 (NED-8), respectively, and do not conjugate Ub (Jones and Candido, 2000; Jones et al., 2002). The functions of the E2 genes have been studied in systematic RNAi screens. Only two of the 20 E2 enzymes that are specific for Ub-conjugation are essential for embryonic viability, ubc-2/let-70 and ubc-14 (Table 1; Jones et al., 2002). This is surprising given the relatively large number of E3 genes that are associated with embryonic lethal phenotypes (see below). This suggests either that UBC-2 and UBC-14 are the only E2s that function with these essential E3s or that there is significant redundancy of E2 function. In general, very little is known about which C. elegans E2s function with particular E3s. Of the remaining Ub-specific E2 genes, the RNAi depletion of four are associated with post-embryonic phenotypes: ubc-19 RNAi produces unhealthy larvae; ubc-20 RNAi produces an impenetrant L3 or L4 larval arrest; ubc-25 RNAi produces defects in neuromuscular function; and ubc-18 RNAi produces animals that have slightly slower growth and reduced brood sizes but otherwise appear wild-type (Maeda et al., 2001; Jones et al., 2002; Schulze et al., 2003; Fay et al., 2003; Table 1). Interestingly, ubc-18 functions redundantly with lin-35 Rb to promote normal pharyngeal morphogenesis, and the simultaneous inactivation of both genes causes synthetic embryonic lethality (Fay et al., 2003). For the remaining 14 E2s, inhibition by RNAi was not associated with any apparent defects.

There are four major classes of ubiquitin ligases: HECT-domain proteins; U-box proteins; monomeric RING finger proteins; and multisubunit complexes that contain a RING finger protein (Passmore and Barford, 2004). HECT-domain E3s are unique in that Ub is transferred to a conserved cysteine residue of the E3 in a thiolester linkage, and then the E3 transfers the Ub to the substrate (Passmore and Barford, 2004; Figure 2). This function as a covalent intermediary in the transfer of Ub is not found in other classes of E3 proteins. The RING finger motif (Really Interesting New Gene) comprises eight cysteine or histidine residues that bind two Zn²⁺ ions in a cross-brace structure (Fang et al., 2003). The U-box is structurally similar to the RING finger motif, but it does not bind Zn²⁺, instead, hydrogen bonds take the place of the Zn²⁺ in the structure (Fang et al., 2003). Monomeric RING finger E3s and U-box E3s bind to both the substrate and the E2 enzyme (Figure 2). In multimeric RING finger complexes, the RING finger protein binds the E2 while other proteins in the complex bind the substrate. These multimeric complexes fall into two classes: cullin-based complexes, and the APC/C (anaphase promoting complex/cyclosome), which contains the cullin-like protein APC2 (Vodermaier, 2004).

Figure 2. A) Structural model of HECT-domain E3 complex. The mechanism for the conjugation of Ub to a substrate by a HECT-domain E3 is shown. The E2 binds the N-terminal lobe of the HECT-domain E3 (top) and transfers Ub to the C-terminal lobe via a thiolester linkage (middle). The C-terminal lobe swivels on a hinge-loop and catalyzes the transfer of Ub to the substrate protein (bottom). B) Model of U-box or monomeric RING finger E3s. The U-box or RING finger domains of the E3 are directly involved in binding the E2. C) Model of SCF complexes. The N-terminus of CUL-1 binds the adaptor Skp1 (SKR proteins in C. elegans), while the C-terminus binds the RING finger protein Rbx1, which binds the E2. The substrate recognition subunit (SRS) binds to Skp1 through an F-box motif. D) Model of CUL-2-based E3 complexes. The N-terminus of CUL-2 binds to the adaptor elongin C (ELC-1), which is in complex with elongin B (ELB-1). The SRS binds to elongin C through a BC-box motif. E) Model of CUL-3-based E3 complexes. The N-terminus of CUL-3 binds directly to the SRS, which utilizes a BTB/POZ domain to bind to CUL-3.