Biology Reference
In-Depth Information
Chapter 45
Assay for Proteasome-Dependent Protein Degradation
and Ubiquitinated Proteins
Takeo Sato , Kaori Sako , and Junji Yamaguchi
Abstract
The ubiquitin-26S proteasome system (UPS) plays a crucial role in selective removal of short-lived target
proteins, archiving fi ne-tuning of post-translation levels of the target proteins. Recently a number of ubiq-
uitin ligases (E3) have been reported as essential regulators of various plant developmental cues and stress
responses. To clarify the detailed biochemical and physiological function of the E3 proteins, identifi cation
of their target proteins is of great importance. A transient expression system with tobacco leaves is a power-
ful method to evaluate E3 function and target degradation via UPS. Here simple methods to assay
proteasome-dependent protein degradation combined with a tobacco transient expression system and
detection of accumulation of ubiquitinated proteins are presented.
Key words
Ubiquitin, Ubiquitin ligase, 26S proteasome, Tobacco,
Agrobacterium
, Transient
expression
1
Introduction
The ubiquitin-26S proteasome system (UPS) controls multiple
phenomena in plant growth and development by regulating the
stability of specifi c key target proteins that govern specifi c cellular
events including photomorphogenesis, cell cycle, senescence,
defense response, and phytohormone response [
1
,
2
]. The ubiqui-
tin molecule attaches to the target protein via E1-E3 enzymes and
the poly-ubiquitinated substrate is then degraded by a multi-
subunit protease complex, the proteasome. Ubiquitin ligase (E3) is
the key enzyme to specify the target protein for degradation via
UPS (Fig.
1
).
The Arabidopsis genome contains more than 1,200 genes
encoding E3 [
3
]. While identifi cation of target proteins is essential
to reveal the details of the function of E3, most targets remain
unclear. Recently, improved proteomics approaches have simplifi ed
the task of fi nding proteins that interact with E3, namely, the can-
didate target proteins. Thus, it is of great importance to evaluate
