1. 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 (May 2017)
DOI: /j.molp
Copyright © 2017 The Author Terms and Conditions

2. 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  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions