Worm Breeder's Gazette 13(5): 55 (February 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Medical Genetics, University of British Columbia, Vancouver, B.C., V6T 1Z3. Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, B.C., V5A 1S6. The kex2/subtilisin proprotein convertase family of enzymes convert biologically inactive precursors into active secreted protein molecules. In C.elegans, the characterization of the bli-4 gene introduces a new member to the kex2/subtilisin family of endoproteases. At least four isoforms of the enzyme blisterase are encoded by bli-4. The D isoform of the bli-4 gene products is structurally most similar to the KEX2 product in yeast. Both carry a transmembrane domain, a feature which distinguishes them from many other convertases. The trafficking signals of these gene products are conserved in a four amino acid motif that is 75% identical between the D product of bli-4 and KEX2. This provides an opportunity to learn about conservation of trafficking and localization signals between species. Based on these similarities of structure, we are testing a cDNA clone of the blisterase D isoform for the ability to rescue a S.cerevisiae strain mutant in the homologous kex2 gene. The cDNA clone has been constructed and inserted into a yeast expression vector, containing the GAL promoter. The resultant construct (pGAL::BLI4D) was transfected into a kex2-deficient yeast strain (Y143- mating type alpha) by LiCl2 treatment. Transformants, produced on selective galactose media, were assayed for expression of the KEX2 homologue, blisterase D. Expression was indicated by formation of a halo around transformant colonies when grown on a lawn of mating factor type-a yeast. Preliminary experiments failed to indicate expression of the blisterase D clone. This may be a result of lack of expression of the bli-4 gene, or the blisterase product may not be transported to the appropriate cellular location. Since the two convertases have only three of the four amino acids in common in the trafficking signal, this fourth amino acid may be critical to the localization of the gene product. Chimeric constructs may help to investigate the critical domain for complementation of the kex2-deficient yeast. Further experimental approaches will include replacing the KEX2 protease domain with bli-4, as well as the carboxy terminus, and investigating whether the KEX2 function can be complemented. If successful, this approach can also be used to generate more mutations in bli-4, in particular temperature-sensitive mutants, since none have thus far been generated by direct C.elegans mutagenesis.