Analysis of F factor TraD membrane topology by use of gene fusions and trypsin-sensitive insertions

This report describes a procedure for characterizing membrane protein topol ogy which combines the analysis of reporter protein hybrids and trypsin-sen sitive 31-amino-acid insertions generated by using transposons ISphoA/in an d ISlacZ/in, Studies of the F factor TraD protein imply that the protein ta kes an a structure with two membrane-spanning sequences and amino and carbo xyl termini facing the cytoplasm. It was possible to assign the subcellular location of one region for which the behavior of fused reporter proteins w as ambiguous, based on the trypsin cleavage behavior of a 31-residue insert ion.