Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system.
The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The beta-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.
Francetic
Olivera
O
Molecular Genetics Unit, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
Buddelmeijer
Nienke
N
Lewenza
Shawn
S
Kumamoto
Carol A
CA
Pugsley
Anthony P
AP
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D036781:Fimbriae Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D021381:Protein Transport; D018271:Signal Recognition Particle / Q000502:physiology*
D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D018271:Signal Recognition Particle; D036781:Fimbriae Proteins