Detection and subcellular localization of mature protein H, involved in excretion of cloacin DF13

Abstract

To investigate whether the pCloDF13 encoded protein H is synthesized as a precursor with a signal sequence and to detect the mature protein H, radioactive labeling experiments with Escherichia coli minicells harboring different pCloDF13 derivatives were carried out. Analysis of the labeled polypeptides on special sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed that the polypeptide encoded by the pCloDF13 gene H is a precursor with an apparent Mr of about 5000. The mature polypeptide had an apparent Mr of about 2800. Localization studies revealed that the mature protein H is predominantly located in the membrane fraction, both in the cytoplasmic and in the outer membranes of the minicells.