Profiling the E. coli Membrane 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 find our dataset very useful for identifying transient and surprisingly novel protein interactions. For example, we discover a trans-periplasmic supercomplex comprising subunits of the Bam and Sec translocons, including membrane-bound chaperones YfgM and PpiD. We identify RcsF and OmpA as bone fide interactors of BamA, and we show that interaction of MetQ with the ABC transporter MetNI depends on its N-terminal lipid anchor. We also discover NlpA as a novel interactor of MetNIQ. 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.

Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis

“…Interestingly, yfgL and yfgM are in the same operon (Blattner et al, 1997); the former encoding a subunit of the BAM complex (BamB) (Wu et al, 2005). Indeed, a recent proteomic analysis of the E. coli 'membrane protein interactome' identifies cross-membrane interactions involving SecYEG, BAM and the chaperones YfgM and PpiD (Carlson et al, 2019). Understanding the interplay of various periplasmic chaperones during OMP passage through the Sec-BAM assembly to the outer membrane will require further attention.…”

Development of a Method Combining Peptidiscs and Proteomics to Identify, Stabilize and Purify a Detergent-Sensitive Membrane Protein Assembly

“…Numerous cytosolic ribosomal subunits are also present in both pulldowns (grey points, Figure 2A and 2B). The detection of numerous protein contaminants is inherent to AP/MS experiments and proteomic analysis in general ( 1, 3, 4 ).…”

“…To circumvent the adverse effects of detergents, several membrane mimetics have been developed to isolate membrane proteins in a water-soluble state (4,(14)(15)(16). Results from our group have shown that the peptidisc enables reconstitution of a significant fraction of the membrane proteome in a form suitable to mass spectrometry analysis (Figure 1) (4,16).…”

“…Detergents must also be carefully removed before mass spectrometry analysis, which tends to decrease the efficiency of protein identification. Second, plasmid-based over-expression of the bait protein often perturbs cell envelope biogenesis and potentially alters the stoichiometry and specificity of protein interactions, leading to adverse effects on cell physiology and complex identification ( 4, 9, 10 ).…”

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

“…The majority of reconstitution methods, however, require significant optimization of the reconstitution conditions and often require additional purification steps, which decrease the final yield 7,8 . The peptidisc spontaneously adapts to the target membrane protein and comparatively, requires little optimization and downstream purification 9,10 . In this protocol, PeptiQuick is presented as a simple means to streamline the reconstitution protocol for downstream protein-protein interaction analysis.…”