MEMBRANE TOPOLOGY OF THE ESCHERICHIA-COLI TOLR PROTEIN REQUIRED FOR CELL-ENVELOPE INTEGRITY

TolR is a 142-amino-acid protein required for the import of colicins a nd bacteriophage and for maintenance of cell envelope integrity. The t opology of TolR in the inner membrane was analyzed by two methods. Fir st, bacteria expressing a series of TolR-beta-galactosidase, TolR-alka line phosphatase, and TolR-beta-lactamase fusions were assayed for the appropriate enzymatic activity. Second, the accessibility of TolR to proteinase K was determined in permeabilized cells and everted vesicle s with an antibody elicited against the carboxyl-terminal 70% of TolR. The results are consistent with TolR spanning the inner membrane once via residues 23 to 43 and with the carboxyl-terminal moiety being exp osed to the periplasm. Quantitative studies with the anti-TolR antibod y indicated the presence of 2 x 10(3) to 3 x 10(3) TolR molecules per cell.