Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE.
We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.
Possot
O M
OM
Unité de Génétique Moléculaire, CNRS, URA 1773, Institut Pasteur, 75724 Paris, Cedex 15, France.
Vignon
G
G
Bomchil
N
N
Ebel
F
F
Pugsley
A P
AP
D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004795:Enzyme Stability; D004885:Erwinia / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000378:metabolism*; D007709:Klebsiella / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D005810:Multigene Family; D010957:Plasmids; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C083615:pulG protein, Klebsiella pneumoniae; C061378:PulC protein, Klebsiella pneumoniae; C072863:PulE protein, E coli; D006026:Glycoside Hydrolases; C100181:pullulanase