Subcellular Localization of Transiently Expressed Fluorescent Fusion Proteins

Abstract

The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system.

Molecular cloning and characterization of the cinnamate 4‐hydroxylase gene from Eupatorium adenophorum

“…The expression of GFP‐tagged EaCA4H was controlled by the cauliflower mosaic virus (CaMV) 35S promoter. The plasmid was introduced into onion epidermal cells by using the particle bombardment method (Collings ). The expression of EaCA4H–GFP was observed 16 h after transformation by confocal microscopy.…”

“…by using the particle bombardment method (Collings 2013). The expression of EaCA4H-GFP was observed 16 h after transformation by confocal microscopy.…”

A strategy for cell-based multiplex diagnostics of Myasthenia gravis and autoimmune encephalitis by modifying the subcellular localization of cell membrane autoantigens

“…shown for Golgi-localized proteins [43]. Many researchers use proteins fused to GFP or RFP reporters to visualize their intracellular traffic directly [3]. An alternative is to target fluorescence reporter proteins to defined compartments by fusion with intracellular localization signals obtained from proteins of e.g.…”

Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants

“…A comprehensive set of fluorescent organelle markers is available in plants (20, 21); they provide excellent tools to confirm the subcellular localization of any proteins. In addition to these in vivo protein markers, several organelle-specific fluorescent dyes have been demonstrated to be useful to determine the subcellular localization of proteins (22). …”