Chapter 16. Fusion reporter approaches to monitor transmembrane helix interactions in bacterial membranes Running head: protein-protein interaction in the membrane

In transenvelope multiprotein machines such as bacterial secretion systems, protein-protein interactions not only occur between soluble domains but are also mediated by helix-helix contacts in the inner membrane. Here, we describe genetic assays commonly used to test interactions between transmembrane α-helices in their native membrane environment. These assays are based on the reconstitution of dimeric regulators allowing the expression of reporter genes. We provide detailed protocols for the TOXCAT and GALLEX assays used to monitor homotypic and heterotypic transmembrane helix-helix interactions.

[1]  C. Cambillau,et al.  Priming and polymerization of a bacterial contractile tail structure , 2016, Nature.

[2]  E. Cascales,et al.  The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization , 2015, PLoS genetics.

[3]  E. Bouveret,et al.  Evidence for new homotypic and heterotypic interactions between transmembrane helices of proteins involved in receptor tyrosine kinase and neuropilin signaling. , 2014, Journal of molecular biology.

[4]  V. Shevchik,et al.  Substrate recognition by the bacterial type II secretion system: more than a simple interaction , 2014, Molecular microbiology.

[5]  V. Shevchik,et al.  Dynamic Interplay between the Periplasmic and Transmembrane Domains of GspL and GspM in the Type II Secretion System , 2013, PloS one.

[6]  F. Goñi,et al.  The transmembrane domain of the T4SS coupling protein TrwB and its role in protein-protein interactions. , 2013, Biochimica et biophysica acta.

[7]  C. Cambillau,et al.  TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System* , 2013, The Journal of Biological Chemistry.

[8]  P. Christie,et al.  A Putative Transmembrane Leucine Zipper of Agrobacterium VirB10 Is Essential for T-Pilus Biogenesis but Not Type IV Secretion , 2013, Journal of bacteriology.

[9]  L. J. Mota,et al.  Identification of Novel Type III Secretion Chaperone-Substrate Complexes of Chlamydia trachomatis , 2013, PloS one.

[10]  E. Bouveret,et al.  The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. , 2012, Methods.

[11]  D. Ladant,et al.  Large‐scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly , 2012, Molecular microbiology.

[12]  Y. Shai,et al.  Transmembrane domains interactions within the membrane milieu: principles, advances and challenges. , 2012, Biochimica et biophysica acta.

[13]  C. Cambillau,et al.  Structural Characterization and Oligomerization of the TssL Protein, a Component Shared by Bacterial Type VI and Type IVb Secretion Systems* , 2012, The Journal of Biological Chemistry.

[14]  P. Bond,et al.  Minor pseudopilin self‐assembly primes type II secretion pseudopilus elongation , 2012, The EMBO journal.

[15]  H. Yin,et al.  Transmembrane domain oligomerization propensity determined by ToxR assay. , 2011, Journal of visualized experiments : JoVE.

[16]  C. Baron,et al.  Quantitative analysis of VirB8-VirB9-VirB10 interactions provides a dynamic model of type IV secretion system core complex assembly. , 2010, Biochemistry.

[17]  R. Lloubès,et al.  The SciZ protein anchors the enteroaggregative Escherichia coli Type VI secretion system to the cell wall , 2010, Molecular microbiology.

[18]  E. Lai,et al.  An IcmF Family Protein, ImpLM, Is an Integral Inner Membrane Protein Interacting with ImpKL, and Its Walker A Motif Is Required for Type VI Secretion System-Mediated Hcp Secretion in Agrobacterium tumefaciens , 2009, Journal of bacteriology.

[19]  Stephanie Unterreitmeier,et al.  An extended ToxR POSSYCCAT system for positive and negative selection of self-interacting transmembrane domains. , 2007, Journal of microbiological methods.

[20]  D. Schneider,et al.  From interactions of single transmembrane helices to folding of alpha-helical membrane proteins: analyzing transmembrane helix-helix interactions in bacteria. , 2007, Current protein & peptide science.

[21]  D. Langosch,et al.  A ToxR‐based dominant‐negative system to investigate heterotypic transmembrane domain interactions , 2006, Proteins.

[22]  K. Blumenthal,et al.  A modified, dual reporter TOXCAT system for monitoring homodimerization of transmembrane segments of proteins. , 2006, Biochemical and biophysical research communications.

[23]  D. Ladant,et al.  Interaction Network among Escherichia coli Membrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis , 2005, Journal of bacteriology.

[24]  F. de la Cruz,et al.  Conjugative coupling proteins interact with cognate and heterologous VirB10-like proteins while exhibiting specificity for cognate relaxosomes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Donald M. Engelman,et al.  GALLEX, a Measurement of Heterologous Association of Transmembrane Helices in a Biological Membrane* , 2003, The Journal of Biological Chemistry.

[26]  D. Ladant,et al.  Genetic systems for analyzing protein-protein interactions in bacteria. , 2000, Research in microbiology.

[27]  Xue-Rong Zhou,et al.  Self-Assembly of the Agrobacterium tumefaciens VirB11 Traffic ATPase , 2000, Journal of bacteriology.

[28]  Xue-Rong Zhou,et al.  Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP‐binding cassette mutations on the assembly and function of the T‐DNA transporter , 1999, Molecular microbiology.

[29]  D. Engelman,et al.  TOXCAT: a measure of transmembrane helix association in a biological membrane. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. Beckwith,et al.  Lambda repressor N-terminal DNA-binding domain as an assay for protein transmembrane segment interactions in vivo. , 1998, Journal of molecular biology.

[31]  D. Ladant,et al.  A bacterial two-hybrid system based on a reconstituted signal transduction pathway. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[32]  S. Lory,et al.  The XcpR protein of Pseudomonas aeruginosa dimerizes via its N‐terminus , 1997, Molecular microbiology.

[33]  H. Fritz,et al.  Dimerisation of the glycophorin A transmembrane segment in membranes probed with the ToxR transcription activator. , 1996, Journal of molecular biology.

[34]  J. C. Hu.  Repressor fusions as a tool to study protein-protein interactions. , 1995, Structure.

[35]  C. Sanders,et al.  Analyzing oligomerization of individual transmembrane helices and of entire membrane proteins in E. coli: A hitchhiker's guide to GALLEX. , 2013, Methods in molecular biology.

[36]  D. Schneider,et al.  Genetic systems for monitoring interactions of transmembrane domains in bacterial membranes. , 2013, Methods in molecular biology.

[37]  J. Beckwith,et al.  A gene fusion method for assaying interactions of protein transmembrane segments in vivo. , 2000, Methods in enzymology.