bli-4 is a member of the
kex2/subtilisin-like family of proprotein convertases and encodes at least four gene products which share a common serine endoproteinase catalytic domain. In addition to the original blistered, viable allele
e937, thirteen
bli-4 lethal mutants have been isolated and grouped into two classes depending on phenotype and intra-complementation data. All of these mutants arrest development in late embryogenesis. We have previously described the molecular lesions within three lethal alleles and have now been able to map and sequence a further nine point mutations using a heteroduplex PCR technique. Of particular interest from this analysis are two alleles
h199 and
h791 in which the
bli-4 coding region is truncated by stop codons within the N-terminus of the protease domain. As a result, the mutant protein products would not be expected to encode functional proteases. However, when
h199 and
h791 are placed in trans to the
e937 allele, the resulting heterozygotes show decreased penetrance of the blistered phenotype. The decrease in penetrance differs markedly to that observed (almost 100%) for heteroallelic combinations of other class II lethal alleles with
e937. Like other members of the convertase family, the
bli-4 gene products are predicted to be expressed as inactive zymogens which include an N-terminal prodomain which is thought to be required for inhibition of protease activity as well as for proper folding of the nascent polypeptide precursor before its removal by autocatalytic cleavage. The predicted truncated
h199 and
h791 products encode the complete prodomain suggesting that perhaps these adducts may work to aid proper folding of the
bli-4 proteins expressed from the
e937 chromosome. This hypothesis is supported by recent results obtained for the distantly related bacterial subtilisin E protease. We are also examining whether epitope tagged constructs of potential substrate gene products are cleaved by
bli-4 isoforms in transgenic nematodes. Plasmids in which the FLAG epitope has been inserted immediately carboxy-terminal to the R-X-K/R-R cleavage site of the substrate gene product have been constructed, initially using
bli-4 gene products themselves in order to determine which of the three putative autocatalytic cleavage sites are used to remove the prodomain. Stable transgenic lines are currently being established. We will examine whether the epitope tagged
bli-4 products (and putative substrates) are processed at this site by taking advantage of the unique properties afforded by two monoclonal antibodies (mAbs) which recognise the FLAG epitope. mAb-M1 will only recognize and bind if the epitope is exposed at the amino-terminus of the protein. In this case cleavage at the proteolytic site is an absolute prerequisite for antibody binding. The second monoclonal M2 recognizes the epitope no matter where the motif is inserted within the target protein. M2 can therefore be used to monitor expression of the construct since it will recognize both the zymogen and the processed, mature forms of the tagged substrate. Transfer of the transforming DNA carrying the epitope tagged substrate constructs to various mutant strains can be achieved by classical genetic crosses. In this way, processing of the substrate proteins can be examined and compared in wild-type and
bli-4 mutant backgrounds by either Western analysis, immunoprecipitation or indirect-immunofluorescent techniques. The application of M1 in indirect-immunofluorescence on whole mount animals should also help determine the temporal and spatial or tissue specific processing of epitope tagged substrates. Genetic analysis suggests that SQT-3 and DAF-7 may represent candidate target proteins for future examination. This work is supported by the MRC of Canada.