Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins

Abstract

Background: The eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively. There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment. We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. We also present a phylogenetic analysis of UBCs.

Results: The C. elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins. RNAi shows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homolog of yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifier SUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifier Rub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless. RNAi of ubc-20, an ortholog of yeast UBC1, results in a low frequency of arrested larval development. A phylogenetic analysis of C. elegans, Drosophila and human UBCs shows that this protein family can be divided into 18 groups, 13 of which include members from all three species. The activating enzymes and the ubiquitin-like proteins NED-8 and SUMO are required for embryogenesis.

Conclusions: The number of UBC genes appears to increase with developmental complexity, and our results suggest functional overlap in many of these enzymes. The ubiquitin-like proteins NED-8 and SUMO and their corresponding activating enzymes are required for embryogenesis.

Figure 1

ClustalW alignment of the region surrounding the active site of the C. elegans, Drosophila and human UBC proteins. The names of the proteins are color-coded: C. elegans (green), Drosophila (blue), and human (red). Subgroups of UBCs, separated by horizontal lines, have Roman numerals and UBC designations that correspond to branches shown on the dendrogram in Figure 2. The UBC designations (right) for the subgroups indicate the most similar S. cerevisiae UBC. The active-site cysteinyl residue (C) is highlighted in red, and an invariant proline (P, green) and tryptophan (W, yellow) are also indicated. Other features indicated are the UBC tripeptide motif HPN and variants (in yellow and blue) (see text)

Figure 2

Phylogenetic relationship of the UBC proteins of C. elegans (green), Drosophila (blue), and human (red). The dendrogram was prepared using the Phylip package of programs as described in Materials and methods. The major branches are separated by dotted lines and assigned Roman numerals that correspond to the numbering system on Figure 1. Some branches have a UBC label that indicates the most similar S. cerevisiae UBC. C. elegans UEV proteins are indicated with an asterisk.

Figure 3

RNAi phenotypes of C. elegans ubc and related genes. Nematodes were treated with RNAi by the feeding method. (a) Reduced and bent tail spike produced by ubc-9 RNAi. (b) Curled and branched tail spike produced by ubc-12 RNAi. (c,d)ubc-14 RNAi produces a blunt and rounded tail region with a protrusion near or surrounding the anus. Both specimens are young larvae. (e) Normal alae on an untreated adult. (f) Branched alae (arrows) produced by ubc-9 RNAi on an adult specimen. (g) Irregular alae that diverge around a granular deposit induced by ubc-12 RNAi. (h) Typical morphologically abnormal L1 seen with ubc-9 RNAi. (i) Arrested embryogenesis produced by ubc-14 RNAi with advanced development of the pharynx. Most specimens treated with ubc-14 RNAi arrest at an earlier stage, without pharyngeal development. (j) Arrested embryogenesis produced by sumo RNAi. (k) Vulval eversion on sumo RNAi-treated young adult. (l) Everted vulva and egg-laying defect produced by uba-2 RNAi. The scale bars indicate 25 μm. Magnification: (a,b,k) ×400; (c-j) ×1,000; (l) ×200.