Chapter Two - ACEMBLing a Multiprotein Transmembrane Complex: The Functional SecYEG-SecDF-YajC-YidC Holotranslocon Protein Secretase/Insertase
Introduction
In vivo, the bacterial core translocon SecYEG interacts with additional components: YidC and SecDF-YajC, which together form a complex referred to as the holotranslocon (HTL). The accessory subunits are believed to stimulate both post- and co-translational translocation (Arkowitz and Wickner, 1994, Beck et al., 2001, Duong and Wickner, 1997b, Luirink et al., 2005, Scotti et al., 2000, Urbanus et al., 2001); however, their mechanism of action remains largely unknown. The majority of research in the Sec translocation field has focused on the core translocon SecYEG. This heterotrimeric protein-conducting channel has been extensively characterized over the past 20 years, and one of the major advances was the appearance of its first X-ray structure a decade ago (van den Berg et al., 2004). However, the understanding of the role of individual components of the HTL as part of the complex still remains poor. The main approaches taken to investigate the interactions between different subunits within the HTL involve co-immunoprecipitation and cross-linking studies. Interactions involved in the formation of the HTL complex were first reported by Duong and Wickner, who co-immunoprecipitated SecYEG together with SecDF and YajC from digitonin-solubilized membranes (Duong & Wickner, 1997a). It was later discovered that the back then newly characterized membrane protein YidC also copurifies with SecYEG (Scotti et al., 2000). In this study, overexpression of translocase complexes, SecYEG and SecDF-YajC, resulted in a significant elevation of YidC levels, suggesting that YidC is also part of the HTL complex. Moreover, YidC and SecYEG were also shown to copurify during membrane protein insertion (Boy & Koch, 2009). Depletion studies revealed another functional interaction within the HTL complex between SecDF-YajC and one of the subunits of the SecYEG channel, SecG (Kato, Nishiyama, & Tokuda, 2003).
More recently, a thorough cross-linking analysis reported that YidC makes contacts with the lateral gate of SecYEG channel. The interaction is seemingly dynamic upon ribosome and ribosome nascent chain binding (Sachelaru et al., 2013). This study also confirmed interactions between YidC and SecDF-YajC (YidC–SecF interaction was previously reported in Xie, Kiefer, Nagler, Dalbey, & Kuhn, 2006), which however appear nonessential for the YidC-SecY contacts (Sachelaru et al., 2013).
X-ray crystal structures of both SecDF (Tsukazaki et al., 2011) and YidC (Kumazaki et al., 2014) have been solved recently. This structural information has shed light onto the function of these complexes and created a new scope for their analysis. Nevertheless, it is difficult to rationalize the function of the individual structures in isolation; therefore, their role in the context of their physiological partners, as part of the HTL, still remains largely unclear. A thorough biochemical, structural, and functional analysis of the HTL has not been possible yet, due to the lack of an isolated stable complex containing a full complement of its seven subunits. Moreover, the heterogeneous character of the complex and its low copy number in cells render its isolation from native source material exceedingly difficult. In order to overcome this challenge, attempts have been previously made to coreconstitute individual subunits of the HTL together into liposomes after their independent purifications. This strategy, however, has its limitations, as individual subunits might not be in the correct orientation competent for complex formation outside their native membrane environment. Therefore, until recently, efforts in studying the architecture and function of this multiprotein complex assembly have remained unsuccessful.
Purification of protein complexes, and in particular of membrane protein complexes, in the quality and quantity required for detailed molecular level functional analysis remains a considerable challenge to date. Most complexes in cells do not exist in sufficient abundance to allow efficient extraction from native source material. Furthermore, endogenous material is often characterized by considerable heterogeneity, related not only to functional aspects but also owing to the fact that the cell at a given time will be dynamically assembling and degrading the complexes that catalyze cellular function. Recombinant overexpression has made a tremendous impact on studying proteins and their interactions, and heterologous production has largely replaced purification of endogenous material in molecular biology laboratories. Much effort has been expended to develop expression systems that can produce not only individual proteins but also multisubunit complexes, into which most of the proteins necessarily assemble to catalyze cellular function. Powerful expression systems for prokaryotic and eukaryotic hosts are being developed and implemented. For instance, the MultiBac system we developed has been particularly useful to produce high-quality multisubunit complexes that could not be produced in Escherichia coli but rely on eukaryotic expression (Barford et al., 2013, Bieniossek et al., 2012, Fitzgerald et al., 2006).
A recent development in expression and purification of multiprotein complexes (Bieniossek et al., 2009) has presented a new opportunity to construct a single multigene expression plasmid encoding all seven subunits of the HTL. This innovative technology, ACEMBL, constitutes a rapid and versatile tool for production of stable multisubunit complexes in E. coli in high quality and quantity and has been successfully used for expression of a range of samples including multiprotein complexes and nucleic acid/protein assemblies (Bieniossek et al., 2009). Often, balancing expression levels and placing purification tags are critical issues for successful heterologous production of complexes. The ACEMBL concept relies on using DNA assembly strategies that allow to combinatorially permutate the elements required for expressing genes coding for individual components of a complex (Bieniossek et al., 2009). These elements include weak and strong promoters to drive transcription, genes of interest, purification tags, proteolytic cleavage sites, and terminator elements, giving rise to expression cassettes that will lead to producing each subunit of a protein complex at a defined level and in the defined setup, in the coexpression experiment. ACEMBL likewise provides the means to combine several genes into polycistronic cassettes and, in addition, to combine several expression cassettes rapidly into multigene expression cassettes (Bieniossek et al., 2009). The underlying technology was termed “tandem recombineering” (TR) and involves a combination of sequence- and ligation-independent multifragment DNA assembly (Li & Elledge, 2007) in conjunction with DNA fusion catalyzed by the Cre recombinase. Cre is a site-specific recombination enzyme, which combines DNAs containing a specific repeat recognition sequence, LoxP (Bieniossek et al., 2009, Fitzgerald et al., 2006). ACEMBL also comprises an array of custom-designed synthetic plasmid modules called acceptors and donors, which can be conjoined by means of Cre-catalyzed plasmid fusion. ACEMBL was originally developed to enable structural genomics pipelines to perform protein complex structure determination in high throughput, in a parallelized setup relying on robotics (Bieniossek et al., 2009, Trowitzsch et al., 2010, Vijayachandran et al., 2011). However, all steps involved can likewise be performed in manual mode, and the protocols developed for robots are also highly efficient when used manually.
The ACEMBL system was first used to produce a heterohexameric complex SecYEG-SecDF-YidC, consisting of 33 unique transmembrane segments (TMSs), in E. coli as an expression host (Bieniossek et al., 2009). However, as soon as it became clear that YajC is also an integral part of the HTL, we made use of the flexibility of the ACEMBL system, allowing easy modification of multigene constructs, to integrate an additional expression module for producing YajC. This enabled us to successfully produce and purify the complete HTL supercomplex (Schulze et al., 2014). Being able to purify a stable complex containing a full complement of all seven HTL subunits allowed subsequent analysis of its organization and function. For this purpose, protein secretion and membrane protein insertion processes were reconstituted from purified components in vitro, which then provided efficient means to elucidate the activity of the HTL in both post- and co-translational translocation, providing unique insight into the function of this vital multiprotein transmembrane machine.
Section snippets
ACEMBLing the HTL Multiprotein Complex
The ACEMBL system was developed to tackle the challenge of producing multiprotein complexes, in particular transmembrane protein complexes for structural and functional studies (Bieniossek et al., 2009). Because ACEMBL was originally designed as part of a structural genomics pipeline, the protocols and procedures were optimized to be sufficiently robust to function in a robotized, high-throughput environment (Bieniossek et al., 2009, Vijayachandran et al., 2011). Automation requires simple
Purifying the HTL
The pACEMBL-HTL multigene expression plasmids, generated by the TR technique and ACEMBL reagents, were transformed into E. coli C43(DE3) cells as a host to minimize potential toxicity associated with the overexpression of membrane proteins (Miroux & Walker, 1996). Freshly transformed E. coli cells were grown in 2 × YT broth with antibiotics to an OD600 of 0.8. Induction using 1 mM IPTG and 0.2% (w/v) arabinose was followed by further 3 h of incubation in shaker flasks. Denaturing polyacrylamide
Incorporation of translocation complexes in proteoliposomes
With the aim to study protein secretion and membrane protein insertion activities of the HTL, compared to the core translocon SecYEG, which was purified according to published protocols (Gold et al., 2010), purified proteins were first incorporated into phospholipid vesicles to form proteoliposomes (PLs), both in the presence and absence of bacteriorhodopsin (BR), as described previously (Collinson et al., 2001, Schulze et al., 2014).
SecYEG has been previously shown to form dimers in the
Discussion and Conclusions
A stable HTL complex, which comprises the core translocon SecYEG, the membrane protein insertase YidC and the poorly understood additional components SecDF-YajC, has been successfully expressed and purified (Schulze et al., 2014). Until recently, this has presented a virtually insurmountable challenge. Being in a position to overexpress and purify an intact complex with the ACEMBL technology and to reconstitute it, enables a thorough analysis of its activity in both post- and co-translational
Acknowledgments
The authors thank Sir John Walker for the E. coli C43 expression strain, Dr. John Bason for help with reconstitution of bacteriorhodopsin, Dr. Ryan Schulze for important contributions to the early stages of the project and all members of the Schaffitzel, Collinson and Berger laboratories for helpful discussions. J. K. was supported by a doctoral training grant from the BBSRC. C. S. is supported by a European Research Council ERC Starting Grant Award. I. C. acknowledges support by the BBSRC
References (52)
- et al.
Secy protein, a membrane-embedded secretion factor of E. coli, is cleaved by the ompt protease invitro
Biochemical and Biophysical Research Communications
(1990) - et al.
Baculovirus expression: Tackling the complexity challenge
Current Opinion in Structural Biology
(2013) - et al.
MultiBac: Expanding the research toolbox for multiprotein complexes
Trends in Biochemical Sciences
(2012) - et al.
The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation
Cell
(1990) - et al.
Mechanism and hydrophobic forces driving membrane protein insertion of subunit II of cytochrome bo 3 oxidase
Journal of Molecular Biology
(2008) - et al.
Membrane biogenesis of subunit II of cytochrome bo oxidase: Contrasting requirements for insertion of N-terminal and C-terminal domains
Journal of Molecular Biology
(2006) - et al.
The oligomeric state and arrangement of the active bacterial translocon
The Journal of Biological Chemistry
(2011) - et al.
Subunit a of cytochrome o oxidase requires both YidC and SecYEG for membrane insertion
Journal of Biological Chemistry
(2006) - et al.
SecA membrane cycling at SecYEG is driven by distinct ATP binding and hydrolysis events and is regulated by SecD and SecF
Cell
(1995) - et al.
Oligomeric rings of the Sec61p complex induced by ligands required for protein translocation
Cell
(1996)
Depletion of SecDF-YajC causes a decrease in the level of SecG: Implication for their functional interaction
FEBS Letters
Mammalian Sec61 is associated with Sec62 and Sec63
The Journal of Biological Chemistry
Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels
Journal of Molecular Biology
Sec62 protein mediates membrane insertion and orientation of moderately hydrophobic signal anchor proteins in the endoplasmic reticulum (ER)
The Journal of Biological Chemistry
A large conformational change couples the ATP binding site of SecA to the SecY protein channel
Journal of Molecular Biology
YidC occupies the lateral gate of the SecYEG translocon and is sequentially displaced by a nascent membrane protein
The Journal of Biological Chemistry
Delta mu H + and ATP function at different steps of the catalytic cycle of preprotein translocase
Cell
The proton motive force lowers the level of ATP required for the invitro translocation of a secretory protein in Escherichia-coli
Journal of Biological Chemistry
Engineering G protein-coupled receptors to facilitate their structure determination
Current Opinion in Structural Biology
New baculovirus expression tools for recombinant protein complex production
Journal of Structural Biology
Targeting, insertion, and localization of Escherichia coli YidC
Journal of Biological Chemistry
Detection of cross-links between FtsH, YidC, HflK/C suggests a linked role for these proteins in quality control upon insertion of bacterial inner membrane proteins
FEBS Letters
Robots, pipelines, polyproteins: Enabling multiprotein expression in prokaryotic and eukaryotic cells
Journal of Structural Biology
SecD and SecF are required for the proton electrochemical gradient stimulation of preprotein translocation
EMBO Journal
YidC, an assembly site for polytopic Escherichia coli membrane proteins located in immediate proximity to the SecYE translocon and lipids
EMBO Reports
The SecYEG preprotein translocation channel is a conformationally dynamic and dimeric structure
EMBO Journal
Cited by (8)
The role of the N-terminal amphipathic helix in bacterial YidC: Insights from functional studies, the crystal structure and molecular dynamics simulations
2022, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :Depletion of the YidC protein results in cell death due to the defective assembly of energy-transducing membrane complexes [5], this makes the YidC protein an attractive antibiotic target. The YidC chaperone is an integral part of the holo translocon (HTL), which folds and assembles multipass transmembrane proteins, as shown in the low-resolution cryo-EM structure of the SecYEG-SecDFYajC-YidC complex [6–8]. The YidC chaperone facilitates the removal of non-mature membrane protein substrates from the lateral gate of the SecY protein, releasing the substrates into the lipid bilayer [9,10].
Production of Multi-subunit Membrane Protein Complexes
2021, Methods in Molecular BiologyEfficient production of a mature and functional gamma secretase protease
2018, Scientific ReportsMembrane protein insertase YidC in bacteria and archaea
2017, Molecular MicrobiologyMultiprotein complex production in E. coli: The SecYEG-SecDFYajC-YidC holotranslocon
2017, Methods in Molecular Biology
- 1
Equal contribution.