CWLP and PRP940 form plasma-membrane nanodomain complexes with aquaporins, interact with PP2A and contribute to dehydration tolerance

Honig, A.; Eilenberg, H.; Zuther, E.; Avisar, D.; Erster, O.; Eshkol, G.; Stein, H.; Reznik, N.; Schuster, S.; Belausov, E.; Abu-Abied, M.; Hotta, T.; Lee, Y.-R. J.; Liu, B.; Sadot, E.; Koncz, C.; Zilberstein, A.; Fichman, Y.

doi:10.1101/2022.10.20.512913

bioRxiv · 10.1101/2022.10.20.512913

CWLP and PRP940 form plasma-membrane nanodomain complexes with aquaporins, interact with PP2A and contribute to dehydration tolerance

Honig, A.·Eilenberg, H.·Zuther, E.·Avisar, D.·Erster, O.·Eshkol, G.·Stein, H.·Reznik, N.·Schuster, S.·Belausov, E.·Abu-Abied, M.·Hotta, T.·Lee, Y.-R. J.·Liu, B.·Sadot, E.·Koncz, C.·Zilberstein, A.·Fichman, Y.

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Abstract

The C-type hybrid-proline-rich protein (HyPRP) AtCWLP and its homolog AtPRP940 are referred as cell wall (CW)-plasma-membrane (PM) linker proteins, but little is known about their functions. Here we show that N-terminal proline-rich domains of CWLP and PRP940, containing glycosylated hydroxyproline residues, contact the CW, while their C-terminal 8CM domains function as PM-scaffolds. Both proteins are detected in PM nanodomains (PM-ND) and show co-localization and co-immunoprecipitation with aquaporins PIP2;1 and PIP2;7. Inhibition of actin polymerization by latrunculin B promotes CWLP-endosome appearance, while blocking the actomyosin-based transport by a truncated form of myosin XI-K relaxes lateral boundaries of CWLP-PIP2;1 PD-NDs. Mass spectrometry data indicate that CWLP co-purifies with dynamins implicated in fission of endocytic PD-ND invaginations. Lack of co-localization and co-immunoprecipitation with aquaporin-binding flotillin (FLOT2) indicates that CWLP and PRP940 mark a new distinct type of PM-ND. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that CWLP and PRP940 interact with multiple aquaporins and several protein phosphatase PP2A-B regulatory subunits. By preventing irreversible separation of CW and PM, and likely assisting PP2A-mediated dephosphorylation of aquaporins and closure of their water channels, overexpression of CWLP confers tolerance to plasmolysis, dehydration and freezing in Arabidopsis and to water shortage in potato plants. Summary StatementArabidopsis Hybrid-Proline-Rich Proteins CWLP and PRP940 occur in association with dynamins, recruit PP2A protein phosphatases to aquaporin water channels in plasma-membrane (PM) nanodomains and elevate tolerance to cellular dehydration.

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Honig, A., Eilenberg, H., Zuther, E., Avisar, D., Erster, O., Eshkol, G., Stein, H., Reznik, N., Schuster, S., Belausov, E., Abu-Abied, M., Hotta, T., Lee, Y.-R. J., Liu, B., Sadot, E., Koncz, C., Zilberstein, A., Fichman, Y.. 2022-10-21. CWLP and PRP940 form plasma-membrane nanodomain complexes with aquaporins, interact with PP2A and contribute to dehydration tolerance. https://doi.org/10.1101/2022.10.20.512913

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