Biology Reference
In-Depth Information
Fig.
1
Schematic model of protein degradation via ubiquitin-proteasome system. E1; Ubiquitin activating
enzyme, E2; Ubiquitin conjugating enzyme, E3; Ubiquitin ligase. The ubiquitin (Ub) molecule attaches to the
target protein via E1-E3 enzymes, termed the “ubiquitin cascade.” 26S proteasome recognizes the poly-
ubiquitin chain attached to the target protein, which is caught and unfolded by 19S regulatory particle (19S RP)
and degraded by 20S core particle (20S CP). The ubiquitin-26S proteasome system controls cell activities
which are involved in multiple phenomena in plant growth and development
whether a candidate protein is an actual target or not. There are
several points to consider in such an evaluation; direct ubiquitina-
tion of the protein by specifi c E3, proteasome-dependent degrada-
tion and accumulation in mutants that are defi cient in specifi c E3
functions. In vitro ubiquitination analysis is the most general way
to test the direct ubiquitination activity of E3 for the target [
4
-
7
],
although the success of this approach depends on assay conditions
after nature of the E3. Furthermore, the ubiquitination may not be
refl ected physiologically. On the other hand, it takes a long time to
prepare a specifi c antibody or establish transgenic plants expressing
epitope-tagged target protein to investigate in vivo degradation of
the target protein. Transient expression in tobacco leaves is a pow-
erful method with which to expeditiously prepare and analyze deg-
radation of a target protein via UPS [
8
,
9
]. Recently, the
combination of this transient expression and in vitro treatment
with MG132, a proteasome inhibitor, has been commonly used as
a fi rst step to test for UPS-dependent degradation. In this chapter,
the use of these procedures to identify 14-3-3 as a target of plant
ubiquitin ligase ATL31 is reported [
7
,
10
].
In addition, in vivo treatment with MG132 is a convenient
way to evaluate the degradation of the target by proteasome and
analyze the function of each proteasome subunit under various
stress conditions [
11
,
12
]. Here in detail a proteasome-dependent
degradation assay with a tobacco transient expression system is
described, as well as accumulation of ubiquitinated protein with in
vivo MG132 treatment.
