Abstract
Investigation of secretion systems is often critical to understanding the virulence mechanisms of bacterial pathogens. With estimates as high as 30–40% of proteins secreted or localized to the cell envelope, information about the subcellular localization and organization of secretion complexes and identification and functional characterization of their substrates are key steps toward understanding these intricate systems. Here we describe a protocol using fluorescent live-cell imaging of fusion proteins that can provide a powerful tool to potentially examine the localization, assembly, and role of each component in the secretion complex. In addition, we describe protocols for the identification of secreted substrates using 1D SDS-PAGE coupled with nano-liquid chromatography (LC) and tandem mass spectrometry (MS/MS), and isobaric tagging for absolute quantification (iTRAQ) coupled with two-dimensional LC and MS/MS. Both experimental approaches are applicable to any similar study of membrane transport systems.
Tanya L. Johnson and Aleksandra E. Sikora have contributed equally to this work.
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References
Shapiro L, McAdams HH, Losick R (2009) Why and how bacteria localize proteins. Science 326:1225–1228
Lybarger SR, Johnson TL, Gray MD, Sikora AE, Sandkvist M (2009) Docking and assembly of the type II secretion complex of Vibrio cholerae. J Bacteriol 191:3149–3161
Sikora AE, Zielke RA, Lawrence DA, Andrews PC, Sandkvist M (2011) Proteomic analysis of the Vibrio cholerae type II secretome reveals new proteins, including three related serine proteases. J Biol Chem 286:16555–16566
Carlsohn E, Nystrom J, Karlsson H, Svennerholm AM, Nilsson CL (2006) Characterization of the outer membrane protein profile from disease-related Helicobacter pylori isolates by subcellular fractionation and nano-LC FT-ICR MS analysis. J Proteome Res 5:3197–3204
Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S et al (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3:1154–1169
Ow SY, Salim M, Noirel J, Evans C, Rehman I, Wright PC (2009) iTRAQ underestimation in simple and complex mixtures: “the good, the bad and the ugly”. J Proteome Res 8:5347–5355
Noirel J, Ow SY, Sanguinetti G, Jaramillo A, Wright PC (2008) Automated extraction of meaningful pathways from quantitative proteomics data. Brief Funct Genomic Proteomic 7:136–146
Caldwell RB, Lattemann CT (2004) Simple and reliable method to precipitate proteins from bacterial culture supernatant. Appl Environ Microbiol 70:610–612
de Barsy M, Jamet A, Filopon D, Nicolas C, Laloux G, Rual JF, Muller A, Twizere JC, Nkengfac B, Vandenhaute J et al (2011) Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2. Cell Microbiol 13:1044–1058
Le Blastier S, Hamels A, Cabeen M, Schille L, Tilquin F, Dieu M, Raes M, Matroule JY (2010) Phosphate starvation triggers production and secretion of an extracellular lipoprotein in Caulobacter crescentus. PLoS One 5:e14198
Feilmeier BJ, Iseminger G, Schroeder D, Webber H, Phillips GJ (2000) Green fluorescent protein functions as a reporter for protein localization in Escherichia coli. J Bacteriol 182:4068–4076
Aronson DE, Costantini LM, Snapp EL (2011) Superfolder GFP is fluorescent in oxidizing environments when targeted via the Sec translocon. Traffic 12:543–548
Dinh T, Bernhardt TG (2011) Using superfolder green fluorescent protein for periplasmic protein localization studies. J Bacteriol 193:4984–4987
Schnell U, Dijk F, Sjollema KA, Giepmans BN (2012) Immunolabeling artifacts and the need for live-cell imaging. Nat Methods 9:152–158
Griffin BA, Adams SR, Tsien RY (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 281:269–272
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Johnson, T.L., Sikora, A.E., Zielke, R.A., Sandkvist, M. (2013). Fluorescence Microscopy and Proteomics to Investigate Subcellular Localization, Assembly, and Function of the Type II Secretion System. In: Delcour, A. (eds) Bacterial Cell Surfaces. Methods in Molecular Biology, vol 966. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-245-2_10
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DOI: https://doi.org/10.1007/978-1-62703-245-2_10
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