Evidence for functionality of transmembrane domains of predicted non-canonical plant phosphotransmitters

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Abstract

Bioinformatic methods have been used to predict a new subclass of proteins among plant phosphotransmitters involved in the signaling system of multistep phosphorelay. In contrast to the canonical soluble nucleocytosolic forms, the found phosphotransmitter sequences, belonging to a wide range of plant taxa, potentially contain transmembrane domains. This suggests localization of such proteins on cell membranes and, therefore, a different function in signaling than that of canonical phosphotransmitters. We tested the functionality of the transmembrane domain of the phosphotransmitter using the protein of the tea plant Camellia sinensis. Membrane localization of the transiently expressed recombinant phosphotransmitter with this domain was confirmed by microscopy and immunoblotting. Thus, for the first time, experimental evidence was obtained for the existence of membrane-bound plant phosphotransmitters with as yet unknown functions. These data suggest the presence of a non-canonical membrane branch of signal transduction in the multistage phosphotransfer system in plants.

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S. N. Lomin

Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences

Email: savelievaek@ya.ru
Russian Federation, Moscow

E. M. Savelieva

Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences

Author for correspondence.
Email: savelievaek@ya.ru
Russian Federation, Moscow

A. S. Elanskaya

Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences

Email: savelievaek@ya.ru
Russian Federation, Moscow

D. V. Arkhipov

Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences

Email: savelievaek@ya.ru
Russian Federation, Moscow

G. A. Romanov

Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences

Email: savelievaek@ya.ru
Russian Federation, Moscow

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Supplementary files

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2. Fig. 1. Scheme of cytokinin signal transduction, with potential non-canonical membrane branching. Functional domains of proteins are indicated by colored boxes. TM – transmembrane domain (green – experimentally confirmed, turquoise – predicted); Hpt – phosphotransfer domain; other domains as in [3]. Short-AHP3 – short version of AHP3 protein (NP_001318703.1); Long-AHP3 – long version of AHP3 protein (NP_001330682.1); TM(CsHpt)-AHP3 – short version of AHP3 protein with transmembrane domain of phosphotransmitter XP_028076088.1 C. sinensis; tZ – trans-zeatin; Pi – “hot” phosphate.

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3. Fig. 2. Localization of phosphotransmitter proteins fused with the fluorescent protein eGFP in N. benthamiana tobacco cells. Short-AHP3-GFP – short version of the AHP3 protein (NP_001318703.1); Long-AHP3-GFP – long version of AHP3 protein (NP_001330682.1; TM(CsHpt)-AHP3-GFP – short version of AHP3 protein with transmembrane domain of phosphotransmitter XP_028076088.1 C. sinensis; TM(CKI1)-AHP3-GFP – short version of AHP3 protein (NP_001318703.1) with transmembrane domain of histidine kinase CKI1 (NP_182265.1); TM-AHK3-TM-GFP – sensor module of cytokinin receptor AHK3 (NP_564276.1), flanked by two transmembrane domains. Nuclei are indicated by white arrow.

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4. Fig. 3. Immunoblotting with potential transmembrane phosphotransmitters. TM(CKI1)-AHP3-GFP – short version of AHP3 protein (NP_001318703.1) with transmembrane domain of histidine kinase CKI1 (NP_182265.1); Short-AHP3-GFP – short version of AHP3 protein (NP_001318703.1); Long-AHP3-GFP – long version of AHP3 protein (NP_001330682.1; TM(CsHpt)-AHP3-GFP – short version of AHP3 protein with transmembrane domain of phosphotransmitter XP_028076088.1 C. sinensis; Anti-GFP AB – antibodies to GFP protein; C – cytosol fraction; M – membrane fraction.

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Presented by Academician of the RAS S.G. Georgieva


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