Ubiquitin C-terminal hydrolases (UCHs) are presumed to contribute greatly to the ubiquitin system by removing ubiquitin (Ub) from ubiquitinated peptides or amides. This activity is thought to be essential for cytoplasmic protein degradation. C.elegans has four genes coding for UCH;
ubh-1,
ubh-2,
ubh-3, and
ubh-4 . Expression of all the four genes was confirmed by RT-PCR. The
ubh-1,
ubh-2 , and
ubh-3 genes are predicted to be organized into an operon, and the encoded protein UBH-1, UBH-2, and UBH-3 are 37%, 32%, and 34% identical to human UCH-L1 and 40%, 34%, and 35% identical to human UCH-L3, respectively, while UBH-4 is 47% identical to human UCH-L5. UCH-L1 is an abundant neuronal enzyme, whose deficiency has been reported to link with neurodegenerative disorders such as Parkinson disease. UBH-1 and UBH-3 were expressed in E.coli as fusion proteins with a His-tag and purified. We assayed these enzymes using C.elegans UB, NEDD8, and SUMO-1 fused with HSV- and His-tags at the C-termini as substrates. Both UBH-1 and UBH-3 showed C-terminal hydrolase activity toward Ub and NEDD8, but not toward SUMO-1. In order to examine the expression pattern of
ubh-1 , we performed GFP expression analysis using
ubh-1::gfp . GFP fluorescence was observed in some neuronal cells, two of which were identified as HSN neurons that are responsible for egg laying. To reveal the function of
ubh-1 , we analyzed the phenotypes of knockout mutant
ubh-1 (
tm526 ) and found egg laying defect; the total number of eggs laid by
ubh-1 (
tm526 ) was 63% of that by the wild-type animals. Additionally, the defecation cycle of the
ubh-1 (
tm526 ) was found to be longer (av. 60 sec) than that of the wild type animals (av. 45 sec). Thus,
ubh-1 is thought to be important for neuronal functions. In the genetic background of
rrf-3 (
pk1246 ), feeding RNAi of
ubh-4 caused burst of gonad arms (60%) with egg laying defect, and abnormal DTC migration. This result suggests the possibility that
ubh-4 may be required for gonadogenesis.