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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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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