Abstract
β-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent β-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent β-barrel OMPs largely via its N-domain but with β-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent β-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving β-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for β-barrel OMP biogenesis.
Highlights
-barrel outer membrane proteins (OMPs) [2], which function in a variety of biological processes, including cell adhesion, envelope organization, nutrient transport, virulence-related protein secretion, and signal transduction [4]
SurA Interacts with -Barrel OMPs via Its N-Domain and with BamA Mainly via Its Satellite P2 Domain—In an attempt to determine the manner in which the multi-domain primary periplasmic chaperone SurA participates in the biogenesis of -barrel OMPs, we employed an in vivo photo-crosslinking approach by individually introducing the unnatural amino acid p-benzoyl-L-phenylalanine (Bpa, a photo-activatable crosslinker) into SurA at 58 selected residue positions spreading among its four domains
A Ternary Complex Involving a -Barrel OMP, SurA, and BamA Is Formed in Living Cells—The respective interaction of the well-separated N-domain and P2 domain of SurA with -barrel OMPs and BamA suggests the formation of a ternary complex involving -barrel OMP, SurA, and BamA. To determine whether such a ternary complex is formed in living cells, we initially designed a dual photo-crosslinking strategy in which Bpa was introduced into SurA simultaneously at two positions, but the dual incorporation of Bpa reduced the expression level of SurA protein to one that was insufficient for performing in vivo photo-crosslinking analysis
Summary
-barrel OMPs [2], which function in a variety of biological processes, including cell adhesion, envelope organization, nutrient transport, virulence-related protein secretion, and signal transduction [4]. Through systematic analyses of the subcellular localization of the protein factors involved in -barrel OMP biogenesis, we revealed the presence of a supercomplex that contains the BamA, BamB, SurA, PpiD the Sec
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call