Figure 6. Western blot for ubiquitin. Panel on the left (anti-Ub) shows the overall level of ubiquitin conjugates from wild-type, spe- 26(it112), or uba-1(it129) young adult hermaphrodites. Equal amounts of soluble protein extracts were detected with ubiquitin-specific mono- clonal antibody. Panel on the right (Coomassie) shows the same extracts stained for total protein. Size standards (MW) are indicated to the far right. doi:10.1371/journal.pgen.1000131.g006 test), there were no gross defects in nuclear or cellular morphology or OMA-1::GFP distribution as embryonic development pro- gressed. Discussion We report here the isolation and characterization of a temperature-sensitive mutation of the uba-1 gene, which encodes the E1 ubiquitin-activating enzyme of C. elegans. Activation by E1 is the first step in the enzymatic pathway that leads to the conjugation of ubiquitin to target proteins. Manipulation of E1 activity by temperature shift provides a mechanism for identifying the many roles for ubiquitination throughout development. Effects of the uba-1(it129) mutation are manifested at both the organismal (i.e., embryonic and larval lethality, reduction in body size) and cellular (sperm-specific sterility) levels, and also result in sex- specific differences of developmental (formation of the male copulatory apparatus) and post-developmental (late-onset male paralysis) processes. The uba-1(it129) mutation causes a substantial UBA-1 in C. elegans Development Table 3. Synthetic interactions between double mutants. Genotype + uba-1(it129) + WT WT mat-3(or180) fzy-1(h1983) WT Mel WT Mel WT, wild type; Mel, maternal embryonic lethality. Data are from a minimum of ten hermaphrodites for each genotype reared at the permissive temperature of 15uC. doi:10.1371/journal.pgen.1000131.t003 reduction of in vivo levels of ubiquitin-conjugated substrates, exhibits synthetic embryonic lethality with components of the anaphase promoting complex (an E3 ubiquitin ligase), and produces delays in early embryonic events known to be regulated by ubiquitin-mediated proteolysis. Taken together, the data indicate that the uba-1(it129) mutation results in a temperature-sensitive reduction in its ubiquitin- activating enzymatic activity. Since the uba-1 gene product is the only E1 enzyme in C. elegans, a reduction in its activity is predicted to negatively impact the function of E2 and E3 enzymes globally. This reduction would extend the half-life of proteins normally targeted for the proteasome, as well as altering the localization and/or activities of other ubiquitin-conjugated substrates. Some of these downstream pathways will be more or less sensitive to a reduction in E1 activity, but the result will be a decrease in the rate of ubiquitination for a wide variety of substrate proteins. In support of this model, Western blotting with anti-ubiquitin antibodies demonstrated an overall reduction in ubiquitin labeling of extracts from the uba-1(it129) mutant strain (Figure 6). Also, structural data from related E1 enzymes predict that the Pro1024Ser mutation in uba-1(it129) might alter its catalytic activity. Finally, the model is consistent with our results in the candidate gene screen (in which reduced levels of UBA-1 by RNAi reproduced both the embryonic lethality and male tail defects) as well as the phenocopy of APC hypomorphic alleles rather than strong loss-of-function mutations (i.e., multicellular vs. one-celled embryonic arrest). An alternative hypothesis, that the uba-1(it129) mutation blocks only one or a few E2/E3 pathways, is less likely. The observed reduction of in vivo ubiquitination in the mutant would require that the bulk of ubiquitin conjugation be mediated by one or a few E3 ligases; however, the hundreds of E3s that are present in C. elegans argue against this model. Furthermore, the range of phenotypes produced by the uba-1 mutation is much broader than those reported for inactivation of any single E2 or E3 enzyme [55], consistent with its participation in multiple E2/E3 pathways. We clearly demonstrate genetic interactions between uba-1(it129) and one E3 pathway, the APC, via synthetic embryonic lethality with mat-3 or fzy-1 alleles. However, the sperm-specific fertilization defect appears to involve a different E3 pathway. This phenotype is not observed in APC mutants but has been reported for mutations in spe-16, which has recently been determined to encode an E3 ubiquitin ligase homolog (Steve L’Hernault, personal communication). Some effects of the uba-1 mutation can be interpreted in light of the variety of phenotypes that arise from the loss of individual E2 or E3 activities. For example, embryonic and larval lethality have been reported for a number of E2 and E3 homologs in large-scale RNAi screens [34–36]. However, the majority of these genes have not been further characterized and, absent additional knowledge of which proteins are substrates for particular E2 and E3 enzymes, PLoS Genetics | www.plosgenetics.org 9 July 2008 | Volume 4 | Issue 7 | e1000131