DnaK promotes the selective export of outer membrane protein precursors in SecA-deficient Escherichia coli.
Consistent with many other results indicating that SecA plays an essential role in the translocation of presecretory proteins across the Escherichia coli inner membrane, we previously found that a approximately 95% depletion of SecA completely blocks the export of periplasmic proteins in vivo. Surprisingly, we found that about 25% of the outer membrane protein (OMP) OmpA synthesized after SecA depletion was gradually translocated across the inner membrane. In this study we analyzed the export of several other OMPs after SecA depletion. We found that 25-50% of each OMP as well as an OmpA-alkaline phosphatase fusion protein was exported from SecA-deficient cells. This partial export was completely abolished by the SecA inhibitor sodium azide and therefore still required the participation of SecA. Examination of a variety of OmpA derivatives, however, ruled out the possibility that OMPs are selectively translocated in SecA-deficient cells because SecA binds to their N termini with unusually high affinity. Export after SecA depletion was observed in cells that lack SecB, the primary targeting factor for OMPs, but was abolished by partial inactivation of DnaK. Furthermore, OmpA could be isolated in a stable complex with DnaK. The data strongly suggest that OMPs require only a relatively low level of translocase activity to cross the inner membrane because they can be preserved in a prolonged export-competent state by DnaK.
Qi
Hai-Yan
HY
Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1810, USA.
Hyndman
Janine B
JB
Bernstein
Harris D
HD
D000251:Adenosine Triphosphatases / Q000235:genetics*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics*; D017353:Gene Deletion; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000378:metabolism; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; C078167:OMPA outer membrane proteins; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli; D000081416:SecA Proteins