Chapter title |
Analysis of Golgi-Mediated Protein Traffic in Plant Cells
|
---|---|
Chapter number | 6 |
Book title |
Plant Protein Secretion
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-7262-3_6 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7261-6, 978-1-4939-7262-3
|
Authors |
Wenjin Shen, Zhidan Xiao, Jinbo Shen, Caiji Gao |
Abstract |
In plant secretory pathways, the Golgi apparatus serves as the major sorting hub to receive de novo synthesized protein from the endoplasmic reticulum for further sorting to post-Golgi compartments or for residence in the cisternae of Golgi stacks. Meanwhile, Golgi functions as a pivotal biochemical factory to make modifications of N-glycans and to produce mature glycoproteins. Fluorescent tag-based confocal microscopy in combination with the brefeldin A drug or the genetic tools to disturb Golgi function have been shown as powerful approaches to analyze Golgi-mediated protein traffic in transiently expressed plant protoplasts or in stably expressed transgenic plants. Various endoglycosidases like Endo H and PNGase F have been widely used to monitor Golgi-mediated glycosylation of secretory proteins. Here, using fluorescently tagged Golgi-resident proteins and known glycosylated proteins as examples, we describe detailed protocols to analyze Golgi-mediated protein traffic and glycosylation in transiently expressed protoplasts derived from Arabidopsis suspension culture cells and in stably expressed transgenic plants. |
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