Topology inversion of SecG is essential for cytosolic SecA-dependent stimulation of protein translocation.

SecG, a subunit of the protein translocon, undergoes a cycle of topology inversion. To further examine the role of this topology inversion, we analyzed the activity of membrane vesicles carrying a SecG-PhoA fusion protein (SecG-PhoA inverted membrane vesicles (IMVs)). In the absence of externally added SecA, SecG-PhoA IMVs were as active in protein translocation as SecG(+) IMVs per SecA. Consistent with this observation, insertion of membrane-bound SecA into SecG-PhoA IMVs was normally observed. On the other hand, externally added SecA did not affect the activity of SecG-PhoA IMVs, but it caused >10-fold stimulation of the translocation activity of SecG(+) IMVs, indicating that the topology inversion of SecG, which cannot occur in SecG-PhoA IMVs, is essential for cytosolic SecA-dependent stimulation of protein translocation. SecG-PhoA IMVs generated a 46-kDa fragment of SecA upon trypsin treatment. The accumulation of this membrane-inserted SecA in the SecG-PhoA IMVs was responsible for the loss of the soluble SecA-dependent stimulation. Moreover, fixation of the inverted SecG topology was found to be dependent on soluble SecA. The dual functions of SecG in protein translocation will be discussed.