USE OF ALKALINE-PHOSPHATASE AS A REPORTER POLYPEPTIDE TO STUDY THE ROLE OF THE SUBTILIN LEADER SEGMENT AND THE SPAT TRANSPORTER IN THE POSTTRANSLATIONAL MODIFICATIONS AND SECRETION OF SUBTILIN IN BACILLUS-SUBTILIS-168

The subtilin leader segment of presubtilin was fused to alkaline phosp hatase (AP), which was used as a reporter polypeptide to study the rol e of the subtilin leader segment in posttranslational modifications du ring the conversion of presubtilin to subtilin and in the translocatio n of presubtilin from the cytoplasm of Bacillus subtilis 168 to the ex tracellular medium, It was observed that the subtilin leader segment c ould be utilized by a wild-type transporter, but secretion was enhance d if the subtilin transporter was available, The subtilin leader was n ot cleaved away from the AP component of the precursor until the precu rsor had been transported to the cell wall, and none of the AP was rel eased into the medium until after cleavage had occurred, The role of S paT, which is an ABC transporter that has been implicated in subtilin secretion, was explored by making a large in-frame deletion from the c entral region of SpaT and observing the effect on translocation of the AP reporter. Instead of having an effect on translocation, the deleti on disrupted proteolytic cleavage of the subtilin leader segment and r elease of the AP reporter into the medium, The AP that was secreted by means of the subtilin leader segment had not undergone any posttransl ational modifications, as assessed by amino acid composition analysis and enzymatic activity analysis.