Abstract
The exocyst, conserved from yeast to plants to mammals, is a hetero-octameric complex that mediates tethering of secretory vesicles to designated sites on the plasma membrane during polarized exocytosis. Because structural studies of the intact exocyst complex have been greatly limited by the low yields of purified proteins, many aspects of the exocyst functions remain poorly understood. Here, we present the protocols for the isolation and purification of the recombinant and the native plant exocyst complex. Given the known diversification of the exocyst subunits in plants, our protocols will likely open the possibility of unraveling the functional significance of these subunits in the context of the fully assembled exocyst complex.
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References
Heider MR, Munson M (2012) Exorcising the exocyst complex. Traffic 13(7):898–907. doi:10.1111/j.1600-0854.2012.01353.x
Sztul E, Lupashin V (2006) Role of tethering factors in secretory membrane traffic. Am J Physiol Cell Physiol 290(1):C11–C26. doi:10.1152/ajpcell.00293.2005
Yu IM, Hughson FM (2010) Tethering factors as organizers of intracellular vesicular traffic. Annu Rev Cell Dev Biol 26:137–156. doi:10.1146/annurev.cellbio.042308.113327
Guo W, Roth D, Walch-Solimena C, Novick P (1999) The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J 18(4):1071–1080. doi:10.1093/emboj/18.4.1071
Cai H, Reinisch K, Ferro-Novick S (2007) Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle. Dev Cell 12(5):671–682. doi:10.1016/j.devcel.2007.04.005
TerBush DR, Maurice T, Roth D, Novick P (1996) The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J 15(23):6483–6494
Sivaram MV, Saporita JA, Furgason ML, Boettcher AJ, Munson M (2005) Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p. Biochemistry 44(16):6302–6311. doi:10.1021/bi048008z
Laufman O, Hong W, Lev S (2013) The COG complex interacts with multiple Golgi SNAREs and enhances fusogenic assembly of SNARE complexes. J Cell Sci 126(Pt 6):1506–1516. doi:10.1242/jcs.122101
Morgera F, Sallah MR, Dubuke ML, Gandhi P, Brewer DN, Carr CM, Munson M (2012) Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1. Mol Biol Cell 23(2):337–346. doi:10.1091/mbc.E11-08-0670
Jin Y, Sultana A, Gandhi P, Franklin E, Hamamoto S, Khan AR, Munson M, Schekman R, Weisman LS (2011) Myosin V transports secretory vesicles via a Rab GTPase cascade and interaction with the exocyst complex. Dev Cell 21(6):1156–1170. doi:10.1016/j.devcel.2011.10.009
Shen D, Yuan H, Hutagalung A, Verma A, Kummel D, Wu X, Reinisch K, McNew JA, Novick P (2013) The synaptobrevin homologue Snc2p recruits the exocyst to secretory vesicles by binding to Sec6p. J Cell Biol 202(3):509–526. doi:10.1083/jcb.201211148
He B, Xi F, Zhang X, Zhang J, Guo W (2007) Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane. EMBO J 26(18):4053–4065. doi:10.1038/sj.emboj.7601834
Zhang X, Orlando K, He B, Xi F, Zhang J, Zajac A, Guo W (2008) Membrane association and functional regulation of Sec3 by phospholipids and Cdc42. J Cell Biol 180(1):145–158. doi:10.1083/jcb.200704128
Baek K, Knodler A, Lee SH, Zhang X, Orlando K, Zhang J, Foskett TJ, Guo W, Dominguez R (2010) Structure-function study of the N-terminal domain of exocyst subunit Sec3. J Biol Chem 285(14):10424–10433. doi:10.1074/jbc.M109.096966
Cole RA, Fowler JE (2006) Polarized growth: maintaining focus on the tip. Curr Opin Plant Biol 9(6):579–588. doi:10.1016/j.pbi.2006.09.014
Zarsky V, Kulich I, Fendrych M, Pecenkova T (2013) Exocyst complexes multiple functions in plant cells secretory pathways. Curr Opin Plant Biol 16(6):726–733. doi:10.1016/j.pbi.2013.10.013
Pecenkova T, Hala M, Kulich I, Kocourkova D, Drdova E, Fendrych M, Toupalova H, Zarsky V (2011) The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plant-pathogen interaction. J Exp Bot 62(6):2107–2116. doi:10.1093/jxb/erq402
Drdova EJ, Synek L, Pecenkova T, Hala M, Kulich I, Fowler JE, Murphy AS, Zarsky V (2013) The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis. Plant J 73(5):709–719. doi:10.1111/tpj.12074
Cvrckova F, Grunt M, Bezvoda R, Hala M, Kulich I, Rawat A, Zarsky V (2012) Evolution of the land plant exocyst complexes. Front Plant Sci 3:159. doi:10.3389/fpls.2012.00159
Wang J, Ding Y, Wang J, Hillmer S, Miao Y, Lo SW, Wang X, Robinson DG, Jiang L (2010) EXPO, an exocyst-positive organelle distinct from multivesicular endosomes and autophagosomes, mediates cytosol to cell wall exocytosis in Arabidopsis and tobacco cells. Plant Cell 22(12):4009–4030. doi:10.1105/tpc.110.080697
Ding Y, Wang J, Chun Lai JH, Ling Chan VH, Wang X, Cai Y, Tan X, Bao Y, Xia J, Robinson DG, Jiang L (2014) Exo70E2 is essential for exocyst subunit recruitment and EXPO formation in both plants and animals. Mol Biol Cell 25(3):412–426. doi:10.1091/mbc.E13-10-0586
Lin Y, Ding Y, Wang J, Shen J, Kung CH, Zhuang X, Cui Y, Yin Z, Xia Y, Lin H, Robinson DG, Jiang L (2015) Exocyst-positive organelles and autophagosomes are distinct organelles in plants. Plant Physiol 169(3):1917–1932. doi:10.1104/pp.15.00953
Munson M, Novick P (2006) The exocyst defrocked, a framework of rods revealed. Nat Struct Mol Biol 13(7):577–581. doi:10.1038/nsmb1097
Zhang C, Brown MQ, van de Ven W, Zhang ZM, Wu B, Young MC, Synek L, Borchardt D, Harrison R, Pan S, Luo N, Huang YM, Ghang YJ, Ung N, Li R, Isley J, Morikis D, Song J, Guo W, Hooley RJ, Chang CE, Yang Z, Zarsky V, Muday GK, Hicks GR, Raikhel NV (2016) Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis. Proc Natl Acad Sci U S A 113(1):E41–E50. doi:10.1073/pnas.1521248112
Hsu SC, Hazuka CD, Roth R, Foletti DL, Heuser J, Scheller RH (1998) Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments. Neuron 20(6):1111–1122
Heider MR, Gu M, Duffy CM, Mirza AM, Marcotte LL, Walls AC, Farrall N, Hakhverdyan Z, Field MC, Rout MP, Frost A, Munson M (2016) Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex. Nat Struct Mol Biol 23(1):59–66. doi:10.1038/nsmb.3146
Hala M, Cole R, Synek L, Drdova E, Pecenkova T, Nordheim A, Lamkemeyer T, Madlung J, Hochholdinger F, Fowler JE, Zarsky V (2008) An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. Plant Cell 20(5):1330–1345. doi:10.1105/tpc.108.059105
Acknowledgments
We thank Liwen Jiang for critical reading of the manuscript. This work was supported by the Direct Grant for Research from the Research Committee of the Chinese University of Hong Kong, China (Project No. 4053182) and Research Grants Council of Hong Kong (14105577, C4012-16E, C4011-14R and AoE/M-05/12).
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Leung, K.P., Lau, W.C.Y. (2017). Isolation of the Plant Exocyst Complex. In: Jiang, L. (eds) Plant Protein Secretion. Methods in Molecular Biology, vol 1662. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7262-3_22
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DOI: https://doi.org/10.1007/978-1-4939-7262-3_22
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