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.