Biology Reference
In-Depth Information
Chapter 31
Isolation of the Plant Cytosolic Fraction for Proteomic
Analysis
Gonzalo M. Estavillo , Yves Verhertbruggen , Henrik V. Scheller,
Barry J. Pogson , Joshua L. Heazlewood , and Jun Ito
Abstract
The cytosol is the fl uid portion of the cell that is not partitioned by membranes. It contains a highly diverse
collection of substances and is central to many essential cellular processes ranging from signal transduction,
metabolite production and transport, protein biosynthesis and degradation to stress response and defense.
Despite its importance, only a few proteomic studies have been performed on the plant cytosol. This is
largely due to diffi culties in isolating relatively pure samples from plant material free of disrupted organelle
material. In this chapter we outline methods for isolating the cytosolic fraction from
Arabidopsis
cell cultures
and seedlings and provide guidance on assessing purity for analysis by mass spectrometry.
Key words
Cytosol, Isolation, Seedling, Cell culture, Arabidopsis, Proteomics
1
Introduction
The plant cytosol is a dynamic and complex intracellular fl uid. It is
the site for numerous biological reactions and often serves as a
channel for metabolic interactions and communication between
membrane-bound organelles. Its aqueous environment is highly
crowded with dissolved ionic solutes, small molecule metabolites,
and macromolecules. Essential processes known to occur in the
plant cytosol include glycolysis [
1
], part of the pentose phosphate
pathway [
2
], protein biosynthesis and degradation [
3
,
4
], signal
transduction [
5
], primary and secondary metabolite biosynthesis
and transportation [
6
], stress response signaling [
7
], and accumu-
lation of enzymes for defense and detoxifi cation [
8
]. It has been
estimated that over 50 % of eukaryotic proteins are present in the
cytosol [
9
].
A large-scale proteomic analysis of cytosolic fractions from
cell suspensions of the model plant,
Arabidopsis thaliana
produced
a set of 1,071 proteins [
10
]. Functional classifi cation of this set
confi rmed many of these proteins have well known cytosol-related
