title: ACEMBLing a multiprotein transmembrane complex: the functional SecYEG-SecDF-YajC-YidC Holotranslocon protein secretase/insertase.
abstract: Membrane proteins constitute about one third of the proteome. The ubiquitous Sec machinery facilitates protein movement across or integration of proteins into the cytoplasmic membrane. In Escherichia coli post- and co-translational targeting pathways converge at the protein-conducting channel, consisting of a central pore, SecYEG, which can recruit accessory domains SecDF-YajC and YidC, to form the holotranslocon (HTL) supercomplex. Detailed analysis of HTL function and architecture remained elusive until recently, largely due to the lack of a purified, recombinant complex. ACEMBL is an advanced DNA recombineering-based expression vector system we developed for producing challenging multiprotein complexes. ACEMBL affords the means to combine multiple expression elements including promoter DNAs, tags, genes of interest, and terminators in a combinatorial manner until optimal multigene expression plasmids are constructed that yield correctly assembled, homogenous, and active multiprotein complex specimens. We utilized ACEMBL for recombinant HTL overproduction. We developed protocols for detergent solubilizing and purifying the HTL. Highly purified complex was then used to reveal HTL function and the interactions between its constituents. HTL activity in protein secretion and membrane protein insertion was analyzed in both the presence and absence of the proton-motive force. Setting up ACEMBL for the assembly of multigene expression constructs that achieve high yields of functional multisubunit membrane protein complex is straightforward. Here, we used ACEMBL for obtaining active HTL supercomplex in high quality and quantity. The concept can likewise be applied to obtain many other assemblies of similar complexity, by overexpression in prokaryotic, and also eukaryotic hosts.
authors: School of Biochemistry, University of Bristol, Bristol, United Kingdom.
authors: Joanna
authors: J
authors: Komar
authors: European Molecular Biology Laboratory, Grenoble, France; Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Unité mixte de Recherche, Grenoble, France.
authors: Mathieu
authors: M
authors: Botte
authors: School of Biochemistry, University of Bristol, Bristol, United Kingdom.
authors: Ian
authors: I
authors: Collinson
authors: School of Biochemistry, University of Bristol, Bristol, United Kingdom; European Molecular Biology Laboratory, Grenoble, France; Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Unité mixte de Recherche, Grenoble, France.
authors: Christiane
authors: C
authors: Schaffitzel
authors: School of Biochemistry, University of Bristol, Bristol, United Kingdom; European Molecular Biology Laboratory, Grenoble, France; Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, Unité mixte de Recherche, Grenoble, France. Electronic address: iberger@embl.fr.
authors: Imre
authors: I
authors: Berger
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D005810:Multigene Family; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D018892:Proton-Motive Force; D000069816:SEC Translocation Channels
chemical_list: D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D021122:Protein Subunits; D000069816:SEC Translocation Channels; C499394:SecD protein, E coli; C412443:YIDC protein, E coli; D000255:Adenosine Triphosphate
keywords: ACEMBL; Combinatorial DNA assembly; Holotranslocon; Membrane protein insertion; Membrane proteins; Multiprotein complex; Protein secretion; Protein-conducting channel; Recombinant production; Translocation