Analysis of Golgi-Mediated Protein Traffic in Plant Cells.

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.