TARGETING OF ENDOPEPTIDASE-24.16 TO DIFFERENT SUBCELLULAR COMPARTMENTS BY ALTERNATIVE PROMOTER USAGE

Endopeptidase 24.16 or mitochondrial oligopeptidase, abbreviated here as EP 24.16 (MOP), is a thiol- and metal-dependent oligopeptidase that is found in multiple intracellular compartments in mammalian cells, F rom an analysis of the corresponding gene, we found that the distribut ion of the enzyme to appropriate subcellular locations is achieved by the use of alternative sites for the initiation of transcription. The pig EP 24.16 (MOP) gene spans over 100 kilobases and is organized into 16 exons, The core protein sequence is encoded by exons 5-16 which ma tch perfectly with exons 2-13 of the gene for endopeptidase 24.15, ano ther member of the thimet oligopeptidase family, These two sets of 11 exons share the same splice sites, suggesting a common ancestor. Multi ple species of mRNA for EP 24.16 (MOP) were detected by the 5'-rapid a mplification of cDNA ends and they were shown to have been generated f rom a single gene by alternative choices of sites for the initiation o f transcription and splicing, Two types of transcript were prepared, c orresponding to transcription from distal and proximal sites, Their ex pression in vit ro in COS-l cells indicated that they encoded two isof orms (long and short) which differed only at their amino termini: the long form contained a cleavable mitochondrial targeting sequence and w as directed to mitochondria; the short form, lacking such a signal seq uence, remained in the cytosol. The complex structure of the EP 24.16 (MOP) gene thus allows, by alternative promoter usage, a fine transcri ptional regulation of coordinate expression, in the different subcellu lar compartments, of the two isoforms arising from a single gene.