Membrane protein insertion and proton-motive-force-dependent secretion through the bacterial holo-translocon SecYEG–SecDF–YajC–YidC

Abstract

Significance In this paper, we describe the biophysical properties, stoichiometry, and activity of the Escherichia coli SecYEG–SecDF–YajC–YidC holo-translocon. This multiprotein complex consists of seven membrane protein subunits, including those components responsible for both protein secretion (SecYEG) and membrane protein insertion (YidC). We demonstrate the isolation of a stable complex containing YidC together with the core SecY translocon. The availability of this intact assembly allows us to reconstitute posttranslational protein export and cotranslational membrane protein insertion from purified components of known stoichiometry. The experiments demonstrate that protein secretion and insertion occur through a single complex. The reconstitution of membrane protein insertion from defined components is a novel development, breaking ground for the functional analysis of this largely unknown process.

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

“…Numerous multisubunit complexes, including soluble multiprotein complexes, protein-RNA complexes and multimeric membrane protein complexes have been produced successfully by ACEMBL [33,44,[60][61][62][63][64]. Examples include the prokaryotic signal recognition particle, SRP, the catalytic cycle of which is being studied by cryo-electron microscopy and biochemical means [60][61][62].…”

“…A particular highlight is the prokaryotic holo-translocon complex (HTL), a seven subunit transmembrane multiprotein assembly consisting of the heterotrimeric core translocon, SecYEG, and its accessory proteins SecD, SecF, YidC and YajC. HTL is a long elusive complex that was, for the first time, successfully produced recombinantly by ACEMBL [63,64]. HTL catalyzes the transport of protein substrates through and into membranes, making use of the proton motive force (PMF) [63].…”

“…HTL is a long elusive complex that was, for the first time, successfully produced recombinantly by ACEMBL [63,64]. HTL catalyzes the transport of protein substrates through and into membranes, making use of the proton motive force (PMF) [63]. Moreover, ACEMBL was applied to reveal the substrate specificity for interferon-stimulated gene 15 by ubiquitin-specific protease 18 [65].…”

Multiprotein Complex Production in E. coli: The SecYEG-SecDFYajC-YidC Holotranslocon

“…Many complexes are characterized by low abundance and heterogeneity in their native hosts, which is refractory to their extraction from native source material, and necessitates recombinant overexpression to purify them in sufficient amounts for detailed mechanistic studies. The SecYEG-SecDFYajC-YidC holotranslocon transmembrane protein complex, HTL, has been discovered in E. coli membranes, where it catalyzes the transport of membrane-bound or periplasmic proteins as they emerge from the actively translating ribosome (3)(4)(5)(12)(13)(14). A particular challenge in expressing functional complexes such as HTL can be encountered when many heterologous subunits are co-produced from individual expression cassettes, resulting in imbalanced expression levels of the individual proteins and impedes proper complex assembly.…”

“…As a consequence, a complex containing all subunits at their physiological stoichiometric ratios cannot be obtained. Moreover, the HTL consists of assembly blocks which by themselves can form stable entities (SecYEG, SecDF and YidC), further complicating HTL production and purification as a homogeneous, functional assembly, prenecessitating elaborate promoter and expression cassette design, tag placement and systematic trial-and-error to achieve success (13)(14)(15). Originally, only six subunits (SecYEG, SecDF and YidC) were thought to be present in HTL and thus included in the overexpression experiments (15).…”