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.
Sugai
Rie
R
Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Takemae
Kazuhisa
K
Tokuda
Hajime
H
Nishiyama
Ken-ichi
K
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001089:Arabinose / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; C088616:SecG protein, E coli; D001089:Arabinose; D014357:Trypsin; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins