Proteasome activity profiling: a simple, robust and versatile method revealing subunit-selective inhibitors and cytoplasmic, defense-induced proteasome activities.
The proteasome plays essential roles in nearly all biological processes in plant defense and development, yet simple methods for displaying proteasome activities in extracts and living tissues are not available to plant science. Here, we introduce an easy and robust method to simultaneously display the activities of all three catalytic proteasome subunits in plant extracts or living plant tissues. The method is based on a membrane-permeable, small-molecule fluorescent probe that irreversibly reacts with the catalytic site of the proteasome catalytic subunits in an activity-dependent manner. Activities can be quantified from fluorescent protein gels and used to study proteasome activities in vitro and in vivo. We demonstrate that proteasome catalytic subunits can be selectively inhibited by aldehyde-based inhibitors, including the notorious caspase-3 inhibitor DEVD. Furthermore, we show that the proteasome activity, but not its abundance, is significantly increased in Arabidopsis upon treatment with benzothiadiazole (BTH). This upregulation of proteasome activity depends on NPR1, and occurs mostly in the cytoplasm. The simplicity, robustness and versatility of this method will make this method widely applicable in plant science.
Gu
Christian
C
Plant Chemetics Lab, Chemical Genomics Centre of the Max Planck Society, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany.
Kolodziejek
Izabella
I
Misas-Villamil
Johana
J
Shindo
Takayuki
T
Colby
Tom
T
Verdoes
Martijn
M
Richau
Kerstin H
KH
Schmidt
Jürgen
J
Overkleeft
Hermen S
HS
van der Hoorn
Renier A L
RA
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology; D001896:Boron Compounds / Q000737:chemistry; D020134:Catalytic Domain; D003593:Cytoplasm / Q000378:metabolism*; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010940:Plant Proteins / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D013379:Substrate Specificity; D013830:Thiadiazoles / Q000494:pharmacology
C517407:Bodipy TMR-Ahx(3)L(3)VS; D001896:Boron Compounds; D005456:Fluorescent Dyes; D009842:Oligopeptides; D010940:Plant Proteins; D011480:Protease Inhibitors; D013830:Thiadiazoles; C015700:benzo-1,2,3-thiadiazole; D046988:Proteasome Endopeptidase Complex