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Untethering the TIR1 auxin receptor from the SCF complex increases its stability and inhibits auxin response

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Abstract

Plant genomes encode large numbers of F-box proteins (FBPs), the substrate recognition subunit of SKP1–CULLIN–F-box (SCF) ubiquitin ligases. There are 700 FBPs in Arabidopsis, most of which are uncharacterized. TIR1 is among the best-studied plant FBPs and functions as a receptor for the plant hormone auxin. Here we use a yeast two-hybrid system to identify novel TIR1 mutants with altered properties. The analysis of these mutants reveals that TIR1 associates with the CULLIN1 (CUL1) subunit of the SCF through the N-terminal H1 helix of the F-box domain. Mutations that untether TIR1 from CUL1 stabilize the FBP and cause auxin resistance and associated growth defects, probably by protecting TIR1 substrates from degradation. Based on these results we propose that TIR1 is subject to autocatalytic degradation when assembled into an SCF. Further, our results suggest a general method for determining the physiological function of uncharacterized FBPs. Finally, we show that a key amino acid variation in the F-box domain of auxin signalling F-box (AFB1), a closely related FBP, reduces its ability to form an SCF, resulting in an increase in AFB1 levels.

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Figure 1: Isolation of TIR1 mutants in yeast.
Figure 2: The tir1E12K and E15K proteins are stable in plants.
Figure 3: The tir1E12K and E15K proteins are deficient in SCF assembly.
Figure 4: The pTIR1:TIR1GUS transgenic lines display severe defects throughout plant growth.
Figure 5: Effects of TIR1 mutations on auxin response.
Figure 6: The H1 region of the F-box domain stabilizes AFB1.

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Acknowledgements

We gratefully acknowledge Eric Klavins for use of the cytometer and Shelly Jang for assistance with calculation of k5 values. This work was supported by the National Institutes of Health (grant no. GM43644, M.E.), the Howard Hughes Medical Institute (M.E.), the Gordon and Betty Moore Foundation (M.E.), and the Paul G. Allen Family Foundation (J.N.).

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H.Y., Y.Z., B.M., B.O.R.B., R.W., J.N. and M.E. conceived and designed experiments. H.Y., Y.Z., B.M., B.O.R.B. and R.W. performed the experiments. M.P. did the phylogenetic analysis. H.Y., Y.Z., B.M., M.P. and M.E. wrote the manuscript.

Corresponding author

Correspondence to Mark Estelle.

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The authors declare no competing financial interests.

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Yu, H., Zhang, Y., Moss, B. et al. Untethering the TIR1 auxin receptor from the SCF complex increases its stability and inhibits auxin response. Nature Plants 1, 14030 (2015). https://doi.org/10.1038/nplants.2014.30

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