Sun, P., Li, S., Lu, D., Williams, J. S. & Kao, T. Pollen S-locus F-box proteins of Petunia involved in S-RNase-based self-incompatibility are themselves subject to ubiquitin-mediated degradation. Plant J. n/a–n/a (2015). doi:10.1111/tpj.12880
S2-SLF1/S3-SLF1/S2-SLF1(CTR) degradation in yeast via UPP
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Y2H no interaction of S2-SLF1 with S2-RNase and S3-RNase
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No S2-SLF1/S3-SLF1/S2-SLF1(CTR) protein in yeast
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UPP in yeast degradation: treatment with MG132
S2-SLF1 degradation in Petunia via UPP
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pollen/in vitro germinated pollen tubes
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S2S3/S2-SLF1-GFP
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cell-free system
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+MG132 +ATP
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GFP level: no degradation of fused GFP
F-box not involved in degradation (not auto-catalytic)
- S2S3/S2-SLF1(CTR) +/- MG132
18-aa degron on C-Terminal of S2-SLF1
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BDM-PUB:predict potential ubiquitination sites (8 Lys)
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8 pGBKT7 with S2-SLF1 truncated to Lys in yeast
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1-191 Detected: Lys192 potential ubiquitination site?
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K192R replacement in after 192 truncate fragments: no increase in stability
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Does 192-328 has a degron?
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S2-SLF1(minus 192-328) is stable
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4 pGBKT7 with S2-SLF1 truncated to Lys between 192 and 329
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narrow down to 295-328
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Lys295 is not ubiquitination site (K295R)
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Divide 295-328 into 295-312/313-328
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GFP:(295-312) vs GDP:(174-191) compared to pBGBKT7-GFP
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295-312 contains a degradation motif
Degron involved in SI function
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S2S3/S2-SLF1(minus 295-312):GFP
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incomplete stability of S2-SLF1 upon deletion of 295-312
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SI not breakdwon in S3 pollens
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Co-IP: S2-SLF1(minus 295-312):GFP interacts with S2-RNase
Discussion
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F-box proteins regulate stability of their substrates (e.g. EBF1/2-EIN3 in ethylene signaling)
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295-312: no known degron motifs
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4 identical aa among all 17 S2-SLFs+3 highly conserved
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Lys295 is not ubiquitination site: outside degron (recongnition sites) ubiquitination sites (e.g. cyclinB and securin- APC/C)
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Serine/Threoine phosphorylation of F-box residues needed for binding, so phosphorylated peptide motis serve as recognition site
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not complete stability/S2-SLF1(1-253) is more unstable than (1-224): additional degron in 225 to 253
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F-box proteins degradation mediated by non-SCF E3 ligase?
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Common degradation of S2-SLF1 in yeast and Petuina: HECT E3 ligase (mono-subunit degradatiob)? UPL1-7 in Arabidopsis
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deletion of 295-312 makes S2-SLF1 not interact with S3-RNase normally
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CTR of SLFs not contain any known motifs in other F-box proteins (WD40): now a degradation motif in CTR
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Dynamic cycling of SLFs is an integral part of SLFs function
Li, S., Sun, P., Williams, J. S. & Kao, T. Identification of the self-incompatibility locus F-box protein-containing complex in Petunia inflata. Plant Reprod. 27, 31–45 (2014).
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SCF complex= Skp1+Cullin 1+Rbx1
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AhSLF-S2 bait in Y2H screen: found AhSSK1 (Q:why in AhSLF-S2 is not degraded in yeast?)
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PhSSK1 is pollen specific (?)
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PhSSK1 as an adaptor in SCF-like complex
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non-canonical SCF complex: S2-SLF1+piCUL1-G+PiSBP1
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all SLFs assembled in same SCF complex?
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pBI101- LAT52P:S2-SLF1:GFP**/S2S3 Co-IP/MS
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S2-SLF1+PiCUL1-P, PiSSK1 and PiRBX1
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pBI101-LAT52P:PiS-SK1:FLAG:GFP/S2S3
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PiRBX1 and PiCUL1-P in the SLF-containing complex
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S2-SLF4/S2-SLFx in similar complex
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PiSSK1 and PiCUL1-P evolved with SLFs?
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SSK1 is a novel class of Skp1-like proteins
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PiCUL1-P is Petunia inflata homolog of SpCUL1 in tomato (Cullin1 in P.inflata are PiCUl1-G and C)
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PiCUl1-P expression is pollen specific, neither PiCUL1-G or C
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PiRBX1 as only conventional SCF complex component in LSF complexs
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If all SLFs formed same complex in pollen specifity, it could be used to see if a candidate SLF is involved in pollen specifity
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complex formed in mature pollens before germination
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PiSBP1(mono-subunit E3) mediate UPP degradatiuon of SLFs not involved in detoxification of S-RNases