Volume 10, Issue 5, Pages 767-770 (May 2017) Rice Chitin Receptor OsCEBiP Is Not a Transmembrane Protein but Targets the Plasma Membrane via a GPI Anchor  Ben-Qiang Gong, Jiao Xue, Nannan Zhang, Lahong Xu, Xinran Yao, Qiu-Jiao Yang, Yang Yu, Hong-Bin Wang, Dandan Zhang, Jian- Feng Li  Molecular Plant  Volume 10, Issue 5, Pages 767-770 (May 2017) DOI: 10.1016/j.molp.2016.12.005 Copyright © 2017 The Author Terms and Conditions

Figure 1 OsCEBiP Is Localized to the Rice Cell Surface via a C-Terminal GPI Anchor. (A) Prediction of the potential transmembrane (TM) sequence and GPI-anchor signal in OsCEBiP and its Arabidopsis homolog AtLYM2. Predicted TM sequences from the program TMpred are marked by green lines and those from DAS-TMfilter by red lines. Putative cleavage sites (the ω sites) for the GPI-anchor modification are indicated by scissors. (B) Schematic diagram of the dual-epitope tagging strategy for OsCEBiP expression. (C) Dual-tagging assays in protoplasts suggest the possibility of membrane targeting of OsCEBiP and AtLYM2 via a GPI anchor. The failure to detect the FLAG tag at the C terminus of OsCEBiP or AtLYM2 indicated the occurrence of C-terminal cleavage during the GPI-anchor modification. The same PVDF membrane was sequentially immunoblotted with anti-HA and anti-FLAG antibodies. GFP-2FLAG was used to indicate the proper operation of immunoblotting when anti-FLAG antibodies were used. (D) Mutations of 12 potential ω sites into inhibitory amino acids (prolines) in OsCEBiP (12P) restore the C-terminal FLAG tag. (E) The OsCEBiP 12P protein dissociates from the membrane fraction labeled by the transmembrane AtCERK1 protein. (F) PI-PLC cleavage assay in protoplasts confirms OsCEBiP as a membrane protein via GPI anchoring. AtCERK1 was used as both a plasma membrane marker and a control indicating that PI-PLC treatment only affects the localization of membrane proteins with a GPI anchor but not those with a transmembrane region. (G) Dual-tagging assays in multiple transgenic rice plants (lines 1–3) expressing OsCEBiP suggest the possibility of membrane targeting of OsCEBiP via a GPI anchor. Note that transgenic line 4 shows no OsCEBiP expression. (H) PI-PLC cleavage assay in transgenic rice confirms OsCEBiP as a membrane protein via GPI anchoring. Membrane total proteins from transgenic rice line 2 were made into two aliquots, one used as input and another used for PI-PLC treatment. Membrane fractions were identified by immunoblotting with anti-H+ ATPase (Arabidopsis) antibodies that have cross-activities to rice H+ ATPases at the plasma membrane. In (C)–(F) and (H), these experiments were repeated at least three times with similar results. Molecular Plant 2017 10, 767-770DOI: (10.1016/j.molp.2016.12.005) Copyright © 2017 The Author Terms and Conditions