Minitags for small molecules: detecting targets of reactive small molecules in living plant tissues using 'click chemistry'.
Small molecules offer unprecedented opportunities for plant research since plants respond to, metabolize, and react with a diverse range of endogenous and exogenous small molecules. Many of these small molecules become covalently attached to proteins. To display these small molecule targets in plants, we introduce a two-step labelling method for minitagged small molecules. Minitags are small chemical moieties (azide or alkyne) that are inert under biological conditions and have little influence on the membrane permeability and specificity of the small molecule. After labelling, proteomes are extracted under denaturing conditions and minitagged proteins are coupled to reporter tags through a 'click chemistry' reaction. We introduce this two-step labelling procedure in plants by studying the well-characterized targets of E-64, a small molecule cysteine protease inhibitor. In contrast to biotinylated E-64, minitagged E-64 efficiently labels vacuolar proteases in vivo. We displayed, purified and identified targets of a minitagged inhibitor that targets the proteasome and cysteine proteases in living plant cells. Chemical interference assays with inhibitors showed that MG132, a frequently used proteasome inhibitor, preferentially inhibits cysteine proteases in vivo. The two-step labelling procedure can be applied on detached leaves, cell cultures, seedlings and other living plant tissues and, when combined with photoreactive groups, can be used to identify targets of herbicides, phytohormones and reactive small molecules selected from chemical genetic screens.
Kaschani
Farnusch
F
Plant Chemetics Lab, Chemical Genomics Centre of the Max Planck Society, Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Cologne, Germany.
Verhelst
Steven H L
SH
van Swieten
Paul F
PF
Verdoes
Martijn
M
Wong
Chung-Sing
CS
Wang
Zheming
Z
Kaiser
Markus
M
Overkleeft
Herman S
HS
Bogyo
Matthew
M
van der Hoorn
Renier A L
RA
D017360:Arabidopsis / Q000737:chemistry*; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D007976:Leupeptins / Q000378:metabolism; D015335:Molecular Probes / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry*; D011480:Protease Inhibitors / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D036226:Seedlings / Q000737:chemistry
D007976:Leupeptins; D015335:Molecular Probes; D011480:Protease Inhibitors; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde