Appendix S1-supplemental methods
Purification of recombinant proteins and cell free degradation assay
Cells (E. coli, BL21) were induced by 0.1 mM IPTG (Gold Biotech) at A600 ~ 0.7, and allowed
to grow for 4 hours at 18°C. Cells were lysed with Cell-lytic B reagent (Sigma), and the clarified
lysates were diluted in binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM
KH2PO4 and 1 mM DTT) supplemented with protease inhibitor cocktail (Sigma) and 1 mM
PMSF. Diluted lysate was loaded onto a glutathione Sepharose 4B column (GE Healthcare
Biosciences). After extensive washing with the binding buffer recombinant proteins were eluted
in 50 mM Tris-Cl (pH 8.8) containing 50 mM reduced glutathione (Sigma) and dialyzed at 4°C
for 4 hrs in a buffer containing 25 mM Tris-Cl (pH 7.5), 10 mM NaCl, 10 mM MgCl2, 10%
glycerol and 1 mM DTT.
One-week-old Arabidopsis seedlings were frozen in liquid nitrogen and crushed in degradation
buffer (25 mM Tris-Cl (pH 7.5), 10 mM NaCl, 10 mM MgCl2, 4 mM PMSF, 5 mM DTT and 10
mM ATP). Protein concentration was determined using Bradford reagent (Bio-Rad). Each
reaction mix contained 500 µg of total protein and 100 ng of recombinant protein. For the
proteasome inhibitor experiments, 200 µM MG132 was added 30 min prior to the experiment.
The reaction mixes were incubated at 22°C, and the reactions were stopped by addition of
SDS-PAGE sample buffer and boiling.
Plasmid construction for protoplast assay
For the protoplast assay, a modified pKYLX80 vector (5XRE)-35Spro:LUC, containing five
tandem repeats of GAL responsive elements (5XRE) followed by a minimal CaMV35S promoter
(−46 to +8 relative to transcription start site), a firefly luciferase gene (LUC) and rbcS
terminator, was used as a reporter (Figure S9 and (Bai et al., 2011; Schardl et al., 1987). The
N-terminal region of UPL3 (UPL31-470) was cloned in a modified pBlueScript (pBS) vector
containing the mirabilis mosaic virus (MMV) promoter (Dey and Maiti 1999), GAL4
DNA-binding domain (amino acids 1–147), and rbcS terminator, to generate plasmid
pBD-UPL31-470. pBD-GL3, pBD-GL3K535R,K536R, pBD-EGL3, and pBD-EGL3K493R,K495R, were
generated by fusion of the corresponding cDNAs to the GAL4 DNA-binding domain under the
MMV promoter and rbcS terminator. Full-length GL3, EGL3, and GL1 cDNAs were cloned
separately into a modified pBS vector, under the CaMV 35S promoter and rbcS terminator, to
generate 35Spro:GL3, 35Spro:EGL3, and 35Spro:GL1, respectively, which were used as effectors in
the protoplast transient assay. A modified pBS plasmid, 35Spro:uidA, containing uidA under the
CaMV 35S promoter and rbcS terminator, was used as an internal control (Figure S9).
Accession Numbers
Sequence data from this article can be found in The Arabidopsis Information Resource
(http://www.arabidopsis.org/): GL3 (At5g41315), EGL3 (At1g63650), UPL3 (At4g38600),
ACT2 (At3g18780) and TUB3 (At5g62700). Arabidopsis T-DNA insertion mutants and
transgenic lines and their identification numbers are SALK_120957C (gl3-4), SALK_077543
(egl3-2) and SAIL_339_F05 (upl3-2). The upl3-2 lines were previously described (Downes et
al., 2003; Kurepa et al., 2008; Kurepa et al., 2009). The gl3 egl3 double mutant is a gift from Dr.
Erich Grotewold, and was previously described (Morohashi et al., 2007).
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