Comparative analysis of expression of two p97 homologues in Caenorhabditis elegans

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Abstract

Caenorhabditis elegans possesses two p97/VCP/Cdc48p homologues, named CDC-48.1 (C06A1.1) and CDC-48.2 (C41C4.8), although their expression regulation and functional diversity have not yet been studied. We therefore investigated spatial and temporal expression patterns of two p97 homologues in this study. RT-PCR and Western blot analysis showed that the amount of cdc-48.1 was about twofold of that of cdc-48.2 in adults and that two p97 homologues were induced by ER stress. The amount of cdc-48.1 mRNA did not increase in the cdc-48.2 deletion mutant and vice versa. In situ hybridization showed that two p97 homologues are mainly expressed in germ cells. In vivo expression analysis by using GFP translational fusion constructs revealed that CDC-48.1::GFP was expressed from embryos through to adult worms, while CDC-48.2::GFP was expressed mainly in embryos. These results suggest that the expression of two p97 homologues of C. elegans is differently regulated and independent of each other.

Section snippets

Materials and methods

Strains of C. elegans and culture condition. Wild-type Bristol N2 strain and mutants, cdc-48.1 (tm544) and cdc-48.2 (tm659), of C. elegans were cultured at 20 °C as described by Brenner [17].

Stress treatment. Worms were treated at 35 °C for 3 h (heat stress) or at 10 °C for 6 h (cold stress). For the starvation treatment, worms were transferred to NGM plates without E. coli for 3 h at 20 °C. Oxidative stress was tested on NGM plates with 100 mM paraquat for 2 h. For ER stress, worms grown on NGM plates

Two p97 homologues of C. elegans

Although mammals and yeast have a single p97 homologue, C. elegans possesses two p97 homologues CDC-48.1 (C06A1.1) and CDC-48.2 (C41C4.8) consisting of 809 and 810 amino acid residues, respectively [16]. These amino acid sequences showed 88.2% identity over the entire proteins. The major tyrosine phosphorylation site was conserved in both CDC-48.1 and CDC-48.2 (Tyr808 of CDC-48.1 and Tyr809 of CDC-48.2) [20]. CDC-48.1 and CDC-48.2 exhibited high degrees of overall similarity with other p97

Conclusion

The results presented here demonstrate that levels and patterns of expression of two p97 homologues of C. elegans are differently regulated. First, the amount of CDC-48.1 is almost double to that of CDC-48.2 in C. elegans (Figs. 2A and C). Second, CDC-48.1 is expressed ubiquitously through all developmental stages, while CDC-48.2 is mainly expressed at embryonic stage (Fig. 4). Third, CDC-48.2 was increased by both 3 mM and 10 mM DTT, while CDC-48.1 was increased only by the 3 mM DTT treatment (

Acknowledgments

We thank Dr. S. Mitani (Tokyo Women’s Medical School) for C. elegans strains and Dr. A. Fire (Stanford University) for GFP expression vectors. We also thank Ms. C. Ichinose and Y. Kawata for secretarial assistance. This work was supported in part by grants from the Ministry of Education, Culture, Science, Sports and Technology, Japan.

References (29)

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