Abstract
Vacuolar proteins are synthesized as precursor forms in the endoplasmic reticulum and are sorted to the vacuole. In this chapter, we introduce two easy methods for the evaluation of vacuolar protein transport using Arabidopsis seeds. These methods are adequate to detect defects in vacuolar transport mediated by endosomes and other trafficking pathways as well. They include an immunoblot assay that monitors the abnormal accumulation of storage protein precursors, and an immunogold labeling assay that monitors the abnormal secretion of storage proteins. Each method facilitates the rapid identification of defects in the transport of endogenous vacuolar proteins in Arabidopsis mutants.
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References
Neuhaus JM, Martinoia E (2011) Plant vacuoles, eLS. Wiley, Chichester, UK. doi:10.1002/9780470015902.a0001675.pub2
Shitan N, Yazaki K (2013) New insights into the transport mechanisms in plant vacuoles. Int Rev Cell Mol Biol 305:383–433
Pedrazzini E, Komarova NY, Rentsch D, Vitale A (2013) Traffic routes and signals for the tonoplast. Traffic 14:622–628
Rojas-Pierce M (2013) Targeting of tonoplast proteins to the vacuole. Plant Sci 211:132–136
Xiang L, Etxeberria E, Van den Ende W (2013) Vacuolar protein sorting mechanisms in plants. FEBS J 280:979–993
Pimpl P, Hanton SL, Taylor JP, Pinto-daSilva LL, Denecke J (2003) The GTPase ARF1p controls the sequence-specific vacuolar sorting route to the lytic vacuole. Plant Cell 15:1242–1256
Hunter PR, Craddock CP, Di Benedetto S, Roberts LM, Frigerio L (2007) Fluorescent reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar compartment in Arabidopsis cells. Plant Physiol 145: 1371–1382
Wolfenstetter S, Wirsching P, Dotzauer D, Schneider S, Sauer N (2012) Routes to the tonoplast: the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts. Plant Cell 24:215–232
Muntz K (1998) Deposition of storage proteins. Plant Mol Biol 38:77–99
Shimada T, Yamada K, Kataoka M, Nakaune S, Koumoto Y, Kuroyanagi M et al (2003) Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana. J Biol Chem 278:32292–32299
Gruis D, Schulze J, Jung R (2004) Storage protein accumulation in the absence of the vacuolar processing enzyme family of cysteine proteases. Plant Cell 16:270–290
Shimada T, Fuji K, Tamura K, Kondo M, Nishimura M, Hara-Nishimura I (2003) Vacuolar sorting receptor for seed storage proteins in Arabidopsis thaliana. Proc Natl Acad Sci U S A 100:16095–16100
Li L, Shimada T, Takahashi H, Ueda H, Fukao Y, Kondo M et al (2006) MAIGO2 is involved in exit of seed storage proteins from the endoplasmic reticulum in Arabidopsis thaliana. Plant Cell 18:3535–3547
Shimada T, Koumoto Y, Li L, Yamazaki M, Kondo M, Nishimura M et al (2006) AtVPS29, a putative component of a retromer complex, is required for the efficient sorting of seed storage proteins. Plant Cell Physiol 47: 1187–1194
Fuji K, Shimada T, Takahashi H, Tamura K, Koumoto Y, Utsumi S et al (2007) Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds. Plant Cell 19:597–609
Takagi J, Renna L, Takahashi H, Koumoto Y, Tamura K, Stefano G et al (2013) MAIGO5 functions in protein export from Golgi-associated endoplasmic reticulum exit sites in Arabidopsis. Plant Cell 25(11):4658–4675
Acknowledgement
We thank Maki Kondo and Hideyuki Takahashi for technical assistance with the EM protocol. This work was supported by Specially Promoted Research of Grant-in-Aid for Scientific Research to I.H-.N. (22000014).
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Shimada, T., Koumoto, Y., Hara-Nishimura, I. (2014). Evaluation of Defective Endosomal Trafficking to the Vacuole by Monitoring Seed Storage Proteins in Arabidopsis thaliana . In: Otegui, M. (eds) Plant Endosomes. Methods in Molecular Biology, vol 1209. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1420-3_10
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DOI: https://doi.org/10.1007/978-1-4939-1420-3_10
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