Profiling the Escherichia coli membrane protein interactome captured in Peptidisc libraries

Abstract

Protein-correlation-profiling (PCP), in combination with quantitative proteomics, has emerged as a high-throughput method for the rapid identification of dynamic protein complexes in native conditions. While PCP has been successfully applied to soluble proteomes, characterization of the membrane interactome has lagged, partly due to the necessary use of detergents to maintain protein solubility. Here, we apply the peptidisc, a ‘one-size fits all’ membrane mimetic, for the capture of the Escherichia coli cell envelope proteome and its high-resolution fractionation in the absence of detergent. Analysis of the SILAC-labeled peptidisc library via PCP allows generation of over 4900 possible binary interactions out of >700,000 random associations. Using well-characterized membrane protein systems such as the SecY translocon, the Bam complex and the MetNI transporter, we demonstrate that our dataset is a useful resource for identifying transient and surprisingly novel protein interactions. For example, we discover a trans-periplasmic supercomplex comprising subunits of the Bam and Sec machineries, including membrane-bound chaperones YfgM and PpiD. We identify RcsF and OmpA as bone fide interactors of BamA, and we show that MetQ association with the ABC transporter MetNI depends on its N-terminal lipid anchor. We also discover NlpA as a novel interactor of MetNI complex. Most of these interactions are largely undetected by standard detergent-based purification. Together, the peptidisc workflow applied to the proteomic field is emerging as a promising novel approach to characterize membrane protein interactions under native expression conditions and without genetic manipulation.

Response to Comment on “Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry”

“…The identity of these complexes is supported by our proteomic studies, native and denaturing gels, tandem MS experiments, and detergent extraction controls. Since publication, new studies supporting many of our findings have emerged, including studies that show cardiolipin mediating binding between the BAM complex and holotranslocon (14), dimeric ANT-1 in reconstituted membranes (15), E. coli ATP synthase bound to SecYEG (16), and relatively low populations of a complex IV dimer, relative to monomer, in amphipols (17).…”

Grad-seq shines light on unrecognized RNA and protein complexes in the model bacteriumEscherichia coli

“…Although there have been several global studies of the protein interactome of E. coli (4)(5)(6)(7)(8)(9)(10)(11)(12)(13), global information about RNA complex formation has been lacking. Our Grad-seq data readily reproduce the major cellular RNPs such as the SRP or RNAP, while also providing information about regulatory RBPs such as CsrA, Hfq and ProQ, which together bind the majority of sRNAs within the cell ( Figure 1F) (84).…”

“…Beyond these binary methods, the E. coli complexome has been studied with global methods directly, i.e., without epitope-tagging or heterologous expression of proteins (9)(10)(11)(12). This includes recent protein-correlation-profiling, whereby complexes of membrane proteins were predicted following the "guilt-by-association" logic after their separation by size exclusion chromatography (13).…”

Structure-function analyses of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation

“…Recently, the dolP gene was connected genetically to the activation of peptidoglycan amidases during E. coli cell division, however this activity has not been directly confirmed experimentally 12 . In contrast, protein interactome studies suggest DolP is a component of the -barrel assembly machine (Bam) complex 13,14 . While these data suggest that DolP may be involved in outer membrane protein (OMP) biogenesis and the regulation of peptidoglycan remodeling, its precise function in either of these processes remained unclear.…”

“…Given that OM permeability defects are often associated with the loss or modification of molecular partners, we sought to identify DolP ligands. Published high-throughput protein:protein interaction data 13,14 suggested that DolP co-located with components of the BAM complex in the OM. Indeed, we observed that simultaneous deletion of dolP and genes coding the non-essential BAM complex components bamB or bamE lead to negative genetic interactions and increased rates of cell lysis (Fig.…”