The Caenorhabditis elegans gene
bli-4 is a complex locus encoding at least four different protein isoforms (Blisterases A, B, C and D), all of which show sequence similarity to a family of enzymes known as the
kex2/subtilisin-like proprotein convertases. This family of enzymes, often referred to as kexins, are increasingly implicated in the proteolytic activation of inactive precursor molecules important in cellular function. Examples of the role of kexins have been identified widely throughout the animal kingdom, from bacterial systems (B. subtilis) to unicellular eukaryotes (S. cerevisiae) and multicellular organisms (Drosophila, mouse, human). In all cases, proteolytic activation of substrate molecules occurs as a result of site-specific cleavage at the carboxy end of paired basic amino acids. The minimal recognition site is also seen within the kexins themselves, suggesting that these are also produced as inactive precursors, subject to autocatalytic activation. Much work is being done in this lab towards understanding the functional role of
bli-4. The specific question addressed here is whether
bli-4 shows evolutionary conservation of function with the kexin members KEX2 (S. cerevisiae) and hfur (human). One approach is to phenotypically rescue yeast KEX2 mutants. A cDNA encoding the blisterase D gene product was cloned into a yeast expression vector, placing it under control of the GAL1 promoter, which expresses at high levels. Blisterase D is predicted to be the most structurally similar to
kex2p. The construct was used to transform KEX2 deletion mutants. Functional complementation is indicated by secretion of processed alpha-mating factor, a substrate of
kex2p. This produces a halo around a patch of transformed cells when plated on a lawn of opposite mating type yeast. Processing of pro-alpha mating factor by Blisterase D was not observed in this experimental system, though the native yeast KEX2 was efficiently expressed. We are now testing whether KEX2 and hfur can functionally replace
bli-4. To address this, germline injection techniques will be used to genetically transform
bli-4 mutants and attempt phenotypic rescue of both viable and lethal mutants. This necessitates the construction of unique vector systems for expressing KEX2 and hfur in C. elegans. These vector systems make use of the endogenous
bli-4 promoter, whose minimal region was defined through lacZ expression experiments. The
bli-4 promoter will ensure that the transgene is expressed appropriately both in developmental time and in tissue location. In addition, each construct contains the 3' untranslated region of
unc-54 (obtained from an A. Fire ectopic expression vector). One of the vector systems also includes the first four exons of
bli-4 which encode the signal sequence and autocatalytic recognition site. The second vector system will not include any coding sequence of
bli-4; thus, any signaling information will be provided by the transgene. These two vectors are currently being used to test the ability of KEX2 (S. cerevisiae) and hfur (human) to functionally rescue
bli-4 mutants. Supported by MRC of Canada.