Mutational analysis of bli-4/kpc-4 reveals critical residues required for proprotein convertase function in C. elegans.
Kex2/subtilisin-like proteinase activity is required for the production of the adult cuticle in the nematode Caenorhabditis elegans. Deletion of the carboxy termini of four of the bli-4/kpc-4 convertase isoforms results in blistering of the adult cuticle. The blisters vary in severity (expressivity) and are not evident in all individuals (reduced penetrance). We have isolated 13 bli-4/kpc-4 mutants that arrest development in late embryogenesis. Using a PCR-based heteroduplex technique, we have identified nucleotide changes responsible for eight of these lethal mutations. The lesions reside within the first 12 exons that are shared by all of the bli-4/kpc-4 gene products, with the majority of mutations clustered within the protease domain. This finding suggests that the protease domain represents a large mutable target. Among these mutations, allele h384 represents a molecular null mutant in which the catalytically essential serine residue (Ser415) is replaced by phenylalanine. Novel missense mutations that change the identity of amino acids evolutionary conserved in all kex2/subtilisin-convertases highlight critical residues essential for activity. We examined the functional activity of BLI-4/KPC-4 products expressed from several lethal mutants by testing their effect on the variable penetrance of blistering exhibited by the e937 allele. We found that the combination of a bli-4/kpc-4 lethal mutation in trans to the bli-4(e937) mutation was sufficient to cause severe blistering in heteroallelic progeny, even in the presence of a known dominant suppressor.
Thacker
C
C
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.
Srayko
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M
Rose
A M
AM
D000483:Alleles; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D005804:Genes, Lethal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009153:Mutagens / Q000633:toxicity; D009154:Mutation; D010641:Phenotype; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D009153:Mutagens; D005020:Ethyl Methanesulfonate; D013381:Subtilisins; C093968:bli-4 protein, C elegans