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A peptidisc-based AP/MS approach to explore the interactome of the bacterial Sec translocon

ABSTRACT: We recently presented the peptidisc membrane mimetic for the global reconstitution of membrane proteomes into water-soluble detergent-free particles, termed peptidisc library. We here develop a method that combines the peptidisc library with affinity pulldown and mass spectrometry (AP/MS) to explore the membrane interactome at native expression level, which minimizes artifacts due to protein overproduction or exposure to detergents. Using the Sec translocon as a case study, we identify an expanded holo-translocon complex, termed the HMD complex, which consists of 9 different membrane subunits - SecYEG, SecDFyajC, YidC, plus YfgM and PpiD. This super-complex can be biochemically isolated in peptidisc but not in detergent. We also detect associations of the HMD complex with the outer membrane Bam complex and we discover a novel unannotated inner membrane protein named YibN. This interactome analysis at native expression level points out the membrane complexes that are dissociated in detergent, yet stable enough to be captured in peptidiscs. We believe this information is critical for the design of biochemical procedures that will enable their successful purification.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Escherichia Coli (strain K12 / W3110 / Atcc 27325 / Dsm 5911)

TISSUE(S): Cell Line Cell

SUBMITTER: Sadhna Phanse

LAB HEAD: Franck Duong

PROVIDER: PXD018866 | Pride | 2023-03-22

REPOSITORIES: pride

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			Action	DRS
	Ecoli_UT_20110328_w3110_ecocyc_v14.1.fasta	Fasta
	HA191023_OT_BM8823-15_CONTROL_FLAG1X_NODETERGENT.raw	Raw
	HA191023_OT_BM8824-15_CONTROL_FLAG1X_NODETERGENT.raw	Raw
	HA191023_OT_BM8825-15_CONTROL_FLAG1X_NODETERGENT.raw	Raw
	HA191023_OT_BM8829-15_PPID_FLAG1X_NODETERGENT.raw	Raw

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