Post-translational modifications of target proteins by small ubiquitin-like modifier (SUMO) proteins modulate many cellular processes in yeast and animals. Here we present the development of monoclonal antibodies (MAb) and polyclonal antibodies (PAb) against Arabidopsis SIZ1 (AtSIZ1) protein with high specificity. Mice were immunized with recombinant AtSIZ1 protein for generating monoclonal antibodies via the classic hybridoma production technique. Anti-AtSIZ1 MAb and PAb were able to detect endogenous AtSIZ1 in Arabidopsis wild type and its complementary line formed by transforming C-siz1-2 mutant with construct containing the AtSIZ1 gene under the control of the native promoter, but not the siz1-2 deletion mutant. These results show that these anti-AtSIZ MAbs are highly sensitive to detect endogenous AtSIZ1 and can be used for immunoblotting and other experimental methods. The new anti-AtSIZ1 MAbs will be essential tools used to investigate the role of AtSIZ1 in plant developmental biology.
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References
1.
VierstraRD, CallisJ. Polypeptide tags, ubiquitous modifiers for plant protein regulation. Plant Mol Biol, 1999; 41:435–442.
2.
MelchiorF. SUMO-nonclasssical ubiquitin. Annu Rev Cell Dev Biol, 2000; 16:591–626.
3.
HayRT. SUMO. A history of modification. Mol Cell, 2005; 18:1–12.
4.
DownesBP, VierstraRD. Post-translational regulation in plants employing a diverse set of polypeptide tags. Biochem Soc Trans, 2005; 33:393–399.
5.
DownesBP, SaraccoSA, LeeSS, CrowellDN, VierstraRD. MUBs, a family of ubiquitin-fold proteins that are plasma membrane-anchored by prenylation. J Biol Chem, 2006; 281:27145–27157.
6.
GillG. Post-translational modification by the small ubiquitin-like modifier SUMO has big effects on transcription factor activity. Curr Opin Genet Dev, 2003; 13:108–113.
7.
SchmidtD, MüllerS. PIAS/SUMO: new partners in transcriptional regulation. Cell Mol Life Sci, 2003; 60:2561–2574.
8.
JohnsonES, GuptaAA. An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell, 2001; 106:735–744.
9.
HeunP. SUMOrganization of the nucleus. Curr Opin Cell Biol, 2007; 19:350–355.
10.
KurepaJ, WalkerJM, SmalleJ, GosinkMM, DavisSJ, DurhamTL, SungDY, VierstraRD. The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and −2 conjugates is increased by stress. J Biol Chem, 2003; 278:6862–6872.
11.
LoisLM, LimaCD, ChuaNH. Small ubiquitin-like modifier modulates abscisic acid signaling in Arabidopsis. Plant Cell, 2003; 15:1347–1359.
12.
MurtasG, ReevesPH, FuYF, BancroftI, DeanC, CouplandG. A nuclear protease required for flowering-time regulation in Arabidopsis reduces the abundance of small ubiquitin-related modifier conjugates. Plant Cell, 2003; 15:2308–2319.
13.
ColbyT, MatthäiA, BoeckelmannA, StuibleHP. SUMO-conjugating and SUMO-deconjugating enzymes from Arabidopsis. Plant Physiol, 2006; 142:318–332.
ChosedR, MukherjeeS, LoisLM, OrthK. Evolution of a signalling system that incorporates both redundancy and diversity: Arabidopsis SUMOylation. Biochem J, 2006; 398:521–529.
19.
YooCY, MiuraK, JinJB, LeeJ, ParkHC, SaltDE, YunDJ, BressanRA, HasegawaPM. SIZ1 small ubiquitin-like modifier E3 ligase facilitates basal thermotolerance in Arabidopsis independent of salicylic acid. Plant Physiol, 2006; 142:1548–1558.
20.
JinJB, JinYH, LeeJ, MiuraK, YooCY, KimWY, Van OostenM, HyunY, SomersDE, LeeI, YunDJ, BressanRA, HasegawaPM. The SUMO E3 ligase, AtSIZ1, regulates flowering by controlling a salicylic acid-mediated floral promotion pathway and through affects on FLC chromatin structure. Plant J, 2008; 53:530–540.
21.
SachdevS, BruhnL, SieberH, PichlerA, MelchiorF, GrosschedlR. PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies. Genes Dev, 2001; 15:3088–3103.
22.
TakahashiY, KahyoT, TohEA, YasudaH, KikuchiY. Yeast Ull1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions as an adaptor between conjugating enzyme and substrates. J Biol Chem, 2001; 276:48973–48977.
23.
NishidaT, YasudaH. PIAS1 and PIASxalpha function as SUMO-E3 ligases toward androgen receptor and repress androgen receptor-dependent transcription. J Biol Chem, 2002; 277:41311–41317.
24.
MintyA, DumontX, KaghadM, CaputD. Covalent modification of p73alpha by SUMO-1. Two-hybrid screening with p73 identifies novel SUMO-1-interacting proteins and a SUMO-1 interaction motif. J Biol Chem, 2000; 275:36316–36323.
25.
HochstrasserM. SP-RING for SUMO. Cell, 2001; 107:5–8.
26.
DuvalD, DuvalG, KedingerC, PochO, BoeufH. The ‘PINIT’ motif, of a newly identified conserved domain of the PIAS protein family, is essential for nuclear retention of PIAS3L. FEBS Lett, 2003; 554:111–118.
27.
OkuboS, HaraF, TsuchidaY, ShimotakaharaS, SuzukiS, HatanakaH, YokoyamaS, TanakaH, YasudaH, ShindoH. NMR structure of the N-terminal domain of SUMO ligase PIAS1 and its interaction with tumor suppressor p53 and A/T-rich DNA oligomers. J Biol Chem, 2004; 279:31455–31461.
28.
TakahashiY, KikuchiY. Yeast PIAS-type Ull1/Siz1 is composed of SUMO ligase and regulatory domains. J Biol Chem, 2005; 280:35822–35828.
29.
SuzukiR, ShindoH, TaseA, KikuchiY, ShimizuM, YamazakiT. Solution structures and DNA binding properties of the N-terminal SAP domains of SUMO E3 ligases from Saccharomyces cerevisiae and Oryza sativa. Proteins, 2008; 75:336–347.
WitzeES, OldWM, ResingKA, AhnNG. Mapping protein post-translational modifications with mass spectrometry. Nat Methods, 2007; 4:798–806.
34.
KageyMH, MelhuishTA, WottonD. The polycomb protein Pc2 is a SUMO E3. Cell, 2003; 113:127–137.
35.
ShiioY, EisenmanRN. Histone sumoylation is associated with transcriptional repression. Proc Natl Acad Sci USA, 2003; 100:13225–13230.
36.
ZhaoX, BlobelG. A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. Proc Natl Acad Sci USA, 2005; 102:4777–4782.
37.
RoscicA, MöllerA, CalzadoMA, RennerF, WimmerVC, GreskoE, LüdiKS, SchmitzML. Phosphorylation-dependent control of Pc2 SUMO E3 ligase activity by its substrate protein HIPK2. Mol Cell, 2006; 24:77–89.
38.
RoscicA, MöllerA, CalzadoMA, RennerF, WimmerVC, GreskoE, LüdiKS, SchmitzML. Phosphorylation-dependent control of Pc2 SUMO E3 ligase activity by its substrate protein HIPK2. Mol Cell, 2006; 24:77–89.
