2-STAGE MODEL FOR INTEGRATION OF THE LYSIS PROTEIN-E OF PHI-X174 INTOTHE CELL-ENVELOPE OF ESCHERICHIA-COLI

As a tool for determining the topology of the small, 91-amino acid Phi X174 lysis protein E within the envelope complex of Escherichia coli, a lysis active fusion of protein E with streptavidin (E-FXa-StrpA) wa s used. The E-FXa-StrpA fusion protein was visualised using immune ele ctron microscopy with gold-conjugated anti-streptavidin antibodies wit hin the envelope complex in different orientations. At the distinct ar eas of lysis characteristic for protein E, the C-terminal end of the f usion protein was detected at the surface of the outer membrane, where as at other areas the C-terminal portion of the protein was located at the cytoplasmic side of the inner membrane. These results suggest tha t a conformational change of protein E is necessary to induce the lysi s process, an assumption supported by proteinase K protection studies. The immune electron microscopic data and the proteinase K accessibili ty studies of the E-FXa-StrA fusion protein were used for the working model of the E-mediated lysis divided into three phases: phase 1 is ch aracterised by integration of protein E into the inner membrane withou t a cytoplasmic status in a conformation with its C-terminal part faci ng the cytoplasmic side; phase 2 is characterised by a conformational change of the protein transferring the C-terminus across the inner mem brane; phase 3 is characterised by a fusion of the inner and outer mem branes and is associated with a transfer of the C-terminal domain of p rotein E towards the surface of the outer membrane of E. coli.