Indecisive M13 procoat protein mutants bind to SecA but do not activate the translocation ATPase

The M13 procoat protein serves as the paradigm for the See-independent memb rane insertion pathway. This protein is inserted into the inner membrane of Escherichia coli with two hydrophobic regions and a central periplasmic lo op region of 20 amino acid residues. Extension of the periplasmic loop regi on renders M13 procoat membrane insertion See-dependent. Loop regions with 118 or more residues required SecA and SecYEG and were efficiently transloc ated in vivo. Two mutants having loop regions of 80 and 100 residues, respe ctively, interacted with SecA but failed to activate the membrane transloca tion ATPase of SecA in vitro. Similarly, a procoat mutant with two addition al glutamyl residues in the loop region showed binding to SecA but did not stimulate the ATPase. The three mutants were also defective for precursor-s timulated binding of SecA to the membrane surface. Remarkably, the mutant p roteins act as competitive inhibitors of the See translocase. This suggests that the region to be translocated is sensed by SecA but the activation of the SecA translocation ATPase is only successful for substrates with a min imum length of the translocated region.