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The function of EHD2 in endocytosis and defense signaling is affected by SUMO

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Abstract

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulates many cellular processes. SUMOylation has been shown to regulate cellular localization and function of a variety of proteins, in some cases affecting nuclear import or export. We have previously characterized two EHDs (EH domain containing proteins) in Arabidospis and showed their involvement in plant endocytosis. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and the leucine rich repeat receptor like protein LeEix2, an effect that requires and intact coiled-coil domain. Inhibition of endocytosis of LeEix2 by EHD2 is effective in inhibiting defense responses mediated by the LeEix2 receptor in response to its ligand EIX. In the present work we demonstrate that SUMOylation of EHD2 appears to be required for EHD2-induced inhibition of LeEix2 endocytosis. Indeed, we found that a mutant form of EHD2, possessing a defective SUMOylation site, has an increased nuclear abundance, can no longer be SUMOylated and is no longer effective in inhibiting LeEix2 endocytosis or defense signaling in response to EIX.

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Acknowledgments

This work was partly supported by the Israel Science Foundation administered by the Israel Academy of Science and Humanities no. 388/12 and by Research Grant Award no. 4312-10 from BARD, The United States–Israel Binational Agriculture Research and Development Fund. FYVE-dsRed was a kind gift from Dr. Jozef Samaj. We thank Drs Mia Horowitz and Olga Pekar for helpful discussion.

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  1. Department of Molecular Biology and Ecology of Plants, Tel-Aviv University, 69978, Tel-Aviv, Israel

    Maya Bar, Silvia Schuster, Meirav Leibman, Ran Ezer & Adi Avni

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  1. Maya Bar
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  2. Silvia Schuster
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  4. Ran Ezer
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Correspondence to Adi Avni.

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11103_2013_148_MOESM1_ESM.tif

Supplemental Figure 1 Interaction between SUMO and AtEHD2. (a) Bimolecular fluorescence complementation (BiFC) visualization of the interaction between SUMO and AtEHD2; SUMO and AtEHD2_K480Q. Fluorescence images of N. benthamiana leaves infiltrated with a mixture of Agrobacterium tumefaciens (OD600 = 0.1) containing Pro35S:YC-SUMO and Pro35S:YN-AtEHD2 or Pro35S:YC-SUMO and Pro35S:YN-AtEHD2_K480Q. Leaf sections were visualized 48 h after injection under a laser-scanning confocal microscope (Zeiss). Bars= 50 μm. (b) Interactions between AtEHD2-K480Q and SUMO in yeast. Yeast cells harboring SUMO (in pBT3-SUC), and AtEHD2-K480Q (in pPR3-N) or appropriate controls as indicated, were grown on galactose medium lacking the amino acids tryptophan, leucine, adenine and histidine containing 15 mM 3-AT and supplemented with X-gal. (TIFF 1,036 kb)

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Bar, M., Schuster, S., Leibman, M. et al. The function of EHD2 in endocytosis and defense signaling is affected by SUMO. Plant Mol Biol 84, 509–518 (2014). https://doi.org/10.1007/s11103-013-0148-7

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