Biology Reference
In-Depth Information
Chapter 6
Identifying Subcellular Protein Localization
with Fluorescent Protein Fusions After Transient
Expression in Onion Epidermal Cells
Andreas Nebenführ
Abstract
Most biochemical functions of plant cells are carried out by proteins which act at very specifi c places within
these cells, for example, within different organelles. Identifying the subcellular localization of proteins is
therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry
out. The discovery of genetically encoded fl uorescent markers has made it possible to tag specifi c proteins
and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use
transient expression of such fl uorescently tagged proteins in onion epidermal cells to determine their sub-
cellular localization relative to known markers.
Key words
Protein localization, Transient expression, Particle bombardment, Fluorescence micros-
copy, Onion epidermis, Organelle markers
1
Introduction
Plant cells carry out a wide variety of functions, ranging from pho-
tosynthesis and basic metabolism over secretion and cytoplasmic
streaming to environmental and pathogen responses. These func-
tions depend on the proper distribution and interaction of a large
number of proteins within the cells. In recent years, it has become
evident that the dynamics of these protein distributions and inter-
actions are essential for their function which makes it imperative to
develop methods to identify these dynamic events in living cells.
Detection of subcellular localization and dynamics of proteins
is usually achieved by creating genetically encoded fl uorescent
derivatives of the proteins of interest, for example, by fusing them
with green fl uorescent protein (GFP, ref. [
1
]). The discovery of red
fl uorescent proteins [
2
] combined with the targeted modifi cation
of fl uorescent protein (FP) genes to create brighter varieties or dif-
ferent colors (e.g., refs. [
3
,
4
]) allows for the direct comparison of
two or more proteins within the same cell, thus greatly facilitating
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