Probing Inner Membrane Protein Topology by Proteolysis
Inner membrane proteins are inserted into the membrane via α-helices. These helices do not only constitute membrane anchors but may mediate specific interactions with membrane protein partners or participate in energetic processes. The number, location, and orientation of these helices is referred to as topology. Bitopic membrane proteins that consist of a single membrane-embedded domain connecting two soluble domains are distinguished from polytopic ones that consist of multiple membrane-spanning helices connected by extramembrane domains. Defining inner membrane protein topology could be achieved by different methods. Here we describe a protease accessibility assay that makes it possible to define topology based on digestion profiles.
Key wordsMembrane protein Inner membrane Insertion Topology Transmembrane segment Bitopic Polytopic Proteolysis Protease Proteinase K Carboxypeptidase Y
Work in EC laboratory is supported by the Centre National de la Recherche Scientifique, the Aix-Marseille Université, and grants from the Agence Nationale de la Recherche (ANR-14-CE14-0006-02 and ANR-15-CE11-0019-01). MSV is a recipient of a doctoral fellowship from the French Ministère de l’Enseignement Supérieur et de la Recherche.
- 2.Bleves S, Lazdunski A, Filloux A (1996) Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas a eruginosa. J Bacteriol 178:4297–4300Google Scholar
- 3.Ross JA, Plano GV (2011) A C-terminal region of Yersinia pestis YscD binds the outer membrane secretin YscC. J Bacteriol 193:2276–2289Google Scholar
- 4.Das A, Xie YH (1998) Construction of transposon Tn3phoA: its application in defining the membrane topology of the A grobacterium tumefaciens DNA transfer proteins. Mol Microbiol 27:405–414Google Scholar
- 5.Vincent MS, Canestrari MJ, Leone P, Stathopulos J, Ize B, Zoued A, Cambillau C, Kellenberger C, Roussel A, Cascales E. (2017) Characterization of the Porphyromonas gingivalis Type IX Secretion trans-envelope PorKLMNP core complex. J Biol Chem. 292:3252–3261.Google Scholar
- 6.Aschtgen MS, Zoued A, Lloubès R, Journet L, Cascales E (2012) The C-tail anchored TssL subunit, an essential protein of the enteroaggregative Escherichia coli Sci-1 type VI secretion system, is inserted by YidC. Microbiology 1:71–82Google Scholar
- 7.Pross E, Soussoula L, Seitl I, Lupo D, Kuhn A (2016) Membrane targeting and insertion of the C-tail protein SciP. J Mol Biol 428:4218–4227.Google Scholar
- 12.Logger L, Aschtgen MS, Guérin M, Cascales E, Durand E (2016) Molecular dissection of the interface between the type VI secretion TssM cytoplasmic domain and the TssG baseplate component. J Mol Biol 428:4424–4437Google Scholar