FIG. 3.

Trypsin treatment of spheroplasts. E. coli JM109 carrying either plasmid pTW42, encoding the TorA-GFOR fusion protein (A), or plasmid pZY470, encoding the wild-type pre-GFOR (B), was labeled with [³⁵S]methionine for 1 min. After a 5-min chase, cells were converted to spheroplasts and divided into three aliquots. Trypsin was added where indicated (+) to digest periplasmic proteins. As a control, cells in one aliquot were disrupted by ultrasonication after trypsin addition. After trypsin treatment, each aliquot was divided into three parts and subjected to immunoprecipitation with antisera against GFOR (lanes 1 to 3), OmpA (lanes 4 to 6), and DnaK (lanes 7 to 9). p, precursor form of TorA-GFOR (A) or pre-GFOR (B); m, mature GFOR. Positions of tryptic fragments are indicated for GFOR (∗), OmpA (+), and DnaK (#). The numbers at the right margin are positions of molecular mass markers.