Volume 4, Issue 5, Pages 854-868 (September 2011) The Rice RMR1 Associates with a Distinct Prevacuolar Compartment for the Protein Storage Vacuole Pathway  Shen Yun , Wang Junqi , Ding Y.u. , Lo Sze Wan , Gouzerh Guillaume , Neuhaus Jean-Marc , Jiang Liwen   Molecular Plant  Volume 4, Issue 5, Pages 854-868 (September 2011) DOI: 10.1093/mp/ssr025 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 The Rice ReMembR-H2 Protein 1 (OsRMR1) and Its Homologs. Alignment of amino acid sequences of the rice RMR1 (ABF94173) used in this study, rice RMR2 (NP_001060777), and the two Arabidopsis RMR proteins, AtRMR1 (AT5G66160) and AtRMR2 (AT1G71980). Gray areas indicate the conserved amino acids sequences among these four RMRs. Dash lines indicate the signal peptides, while the predicted TMDs are underlined. The conserved C3H2C3 RING fingers are highlighted in boxes. The C2 peptide used to generate antibodies (Figure 2) is doubly underlined. Molecular Plant 2011 4, 854-868DOI: (10.1093/mp/ssr025) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 Generation and Characterization of OsRMR1 Antibodies. (A) Predicted structure of OsRMR1 and its antibodies generation. The predicted topology of OsRMR1 consists of a SP, a protease-associated (PA) domain at its N-terminus, a single TMD and a Zinc C3H2C3 RING finger (ZF) at its CT. The numbers below the construct indicate the positions of amino acid residues of each component. Either an E. coli-produced recombinant GST fusion with the N-terminus of OsRMR1 (termed GST–NT) or a synthetic peptide (termed C2 peptide) corresponding to the amino acids 512–529 of OsRMR1 (doubly underlined in Figure 1) were used as antigens to generate OsRMR1a and OsRMR1b antibodies, respectively. (B) E. coli cells transformed with the empty pGEX4T-1 vector (lane 1) or transformed with pGEX4T-1 encoding GST–NT (lanes 2 and 3) were induced with 1 mM IPTG for 4 h, followed by cell harvest and protein extraction for subsequent separation via SDS–PAGE and staining with Coomassie blue. Asterisk indicates the expressed 42-kDa GST–NT fusion proteins. M, molecular mass in kilodaltons. (C) Purified E. coli-derived recombinant GST–NT proteins were separated by SDS–PAGE, followed by either Coomassie blue staining (lane 1) or immunoblot detection using anti-GST (lane 2) or anti-OsRMR1 (lanes 3 and 4) antibodies. Asterisk indicates the detected GST–NT protein. (D) Immunoblot analysis of proteins isolated from various plant cells or tissues as indicated using either anti-OsRMR1b (lanes 1–4) or anti-OsRMR1a (lanes 5–8) antibodies. Asterisk indicates the detected RMR proteins. M, molecular mass in kilodaltons. Molecular Plant 2011 4, 854-868DOI: (10.1093/mp/ssr025) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 ImmunoEM Localization of OsRMR1 in Rice Cultured Cells. Ultra-thin sections prepared from high-pressure frozen/freeze-substituted rice cultured cells were labeled with OsRMR1a and OsRMR1b antibodies, where gold particles were found in Golgi apparatus (g), trans-Golgi network (T), and the distinct organelle containing an electron-dense core and an undefined limiting membrane (panels 1–6, indicated by asterisk). VSRat-1 antibody labeled MVB but not the distinct organelle (panel 6). Arrows in panels 1–6 indicate examples of gold particles on labeled structures. G, Golgi apparatus; MVB, multivesicular body; T, trans-Golgi network. Scale bar = 200 nm. Molecular Plant 2011 4, 854-868DOI: (10.1093/mp/ssr025) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 OsRMR1 Resides in the Golgi Apparatus and in the Distinct Organelle in Rice Developing Seeds. Ultra-thin sections prepared from high-pressure frozen/freeze-substituted rice developing seeds (10 DAF) were labeled with OsRMR1a and OsRMR1b antibodies, and gold particles were found on Golgi apparatus (g) and the distinct organelle (asterisk) containing an electron-dense core and unclear limiting membranes (panels 1–5, indicated by asterisk). In contrast, anti-VSRat-1 labeled MVB but not the distinct organelle (panel 6). Arrows indicate examples of gold particles on labeled structures. G, Golgi apparatus; MVB, multivesicular body; T, trans-Golgi network. Scale bar = 200 nm. Molecular Plant 2011 4, 854-868DOI: (10.1093/mp/ssr025) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 OsRMR1 Accumulates in PB II or PSV in Rice Developing Seeds. Ultra-thin sections prepared from high-pressure frozen/freeze-substituted rice developing seeds at 10 DAF were either double-labeled with both OsRMR1b antibodies (10-nm gold particles) and glutelin antibodies (15-nm gold particles) (panels 1–4) or singly labeled with prolamin antibodies (10-nm gold particles) (panel 5). Arrows and arrowheads indicate examples of OsRMR1b (10-nm gold) and glutelin (15-nm gold) labeling, respectively, where OsRMR1 labeling can only be found in PBII (PSV) along with glutelin. Scale bar = 500 nm. Molecular Plant 2011 4, 854-868DOI: (10.1093/mp/ssr025) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 6 ImmunoEM Localization of OsRMR1 in Tobacco BY-2 Cells. Ultra-thin sections prepared from high-pressure frozen/freeze-substituted WT BY-2 cells were labeled with OsRMR1a and OsRMR1b antibodies, where gold particles were found in Golgi apparatus (g), trans-Golgi network (T), and a distinct organelle containing electron-dense cores and undefined limiting membranes (indicated by asterisk), but absent from the mitochondria (Mt) (panels 1–5). In contrast, anti-VSRat-1 labeled MVB (arrows) but not the distinct organelle (asterisk) (panel 6). Arrows indicate examples of gold particles on labeled structures. Scale bar = 200 nm. Molecular Plant 2011 4, 854-868DOI: (10.1093/mp/ssr025) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 7 ImmunoEM Localization of OsRMR1 in Arabidopsis Culture Cells. (A) Identification of RMR-positive organelles in Arabidopsis suspension cultured cells. Ultra-thin sections prepared from high-pressure freeze/frozen-substituted Arabidopsis culture cells were labeled with OsRMR1a antibodies. Asterisks indicate examples of RMR-positive organelles (panels 1–6). g, Golgi apparatus. Scale bar = 200 nm. (B) Ultra-structural study of various organelles from sub-cellular fractioned organelles of Arabidopsis culture cells. Protoplasts were prepared from Arabidopsis suspension culture cells, followed by sub-cellular fractionation in sucrose density gradient and identification of marker-enriched fractions. Various Spurr-embedded marker-enriched fractions were then subjected to ultra-thin sectioning and subsequent structural and immunogold EM studies. Shown are examples of morphology of oil bodies (G), Mitochondria (H), MVBs (panels 7–10), and the OsRMR1-positive organelles (panels 11–14). Arrows indicate examples of OsRMR1a labeling. Scale bar = 200 nm. Molecular Plant 2011 4, 854-868DOI: (10.1093/mp/ssr025) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 8 In Vivo Interaction between a Vicilin-Like Storage Protein (VLSP) and the Truncated OsRMR1 in Transgenic BY-2 Cells. (A) Chimeric DNA constructs of OsRMR1 NT-T7. T7 tag was fused with the N-terminal region (upstream of TMD) of OsRMR1. The fusion was expressed under the control of the constitutive 35S CaMV promoter and the NOS terminator. (B) In vivo receptor–cargo interaction for cargo identification. Transgenic BY-2 cells expressing the truncated OsRMR1 protein NT-T7 are subjected to medium collection and protein precipitation, followed by protein analysis via LC–MS/MS for subsequent protein identification that may represent the putative OsRMR1 cargo proteins. (C) Detection and analysis of the secreted truncated OsRMR1 protein NT-T7. Day-7 culture media of either WT or transgenic BY-2 cells expressing NT-T7 were collected for protein precipitation and separation via SDS–PAGE, followed by immunoblot detection with either anti-T7 or anti-OsRMR1a antibodies. Asterisk indicates the 18-kDa truncated OsRMR1 NT-T7 fusion proteins. M, molecular mass in kilodaltons. (D) Identification of the secreted proteins. Day-7 cultured media of either WT or transgenic BY-2 cells expressing NT-T7 were collected for protein precipitation and separation via SDS–PAGE, followed by protein detection via silver staining and nano LC–MS/MS analysis of the detected protein bands for subsequent protein identification via database searching. (E) Protein identification of selective protein bands from the culture media via Nano LC–MS/MS analysis and subsequent database searching for protein identification. Shown are the identity of protein bands 3 and 8 with multiple peptides from MS/MS analysis. Molecular Plant 2011 4, 854-868DOI: (10.1093/mp/ssr025) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 9 In Vitro Interactions between the Secreted Truncated OsRMR1 (NT-T7) and VSD-Containing Phaseolin or VLSP Peptides Were Sequence-Specific and pH-Dependent. (A) In vitro binding of OsRMR1 NT-T7 to VSD-containing peptides of phaseolin and VLSP was sequence-specific. 5 ml of day-7 cultured media (pH at 6.0) of transgenic OsRMR1 NT-T7 BY-2 cells were bound with each of the six columns conjugated with 0.3 mg synthetic peptides of aleurain NPIR (N), phaseolin (P), mutated phaseolin (mP), VLSP (V), mutated VLSP1 (mV1), and mutated VLSP2 (mV2) as indicated, followed by protein elution from the columns using pH 4.0 buffer and protein precipitation. Re-suspended proteins were further separated by SDS–PAGE, followed by immunoblot analysis using anti-T7 antibodies, where both total media before binding and after binding (i.e. flow-through media, Total, and FT, respectively) (lanes 1 and 2) were used as positive controls. Asterisk indicates the truncated OsRMR1 NT-T7 proteins detected by T7 antibodies. M, molecular mass in kilodaltons. (B) In vitro binding of truncated OsRMR1 NT-T7 with VSD-containing phaseolin or VLSP peptides was pH-dependent. The culture medium of transgenic OsRMR1 NT-T7 BY-2 cells was collected and its pH was adjusted to either 4.0, 6.0, or 7.0. Media with these three defined pH were then used for binding with the three columns conjugated with peptides of aleurain NPIR (N) or phaseolin (P) or VLSP (V). Proteins eluted from the columns via boiling in SDS loading buffer were subjected to SDS–PAGE and immunoblot analysis using T7 antibodies. Asterisk indicates the truncated OsRMR1 NT-T7 proteins detected by T7 antibodies. M, molecular mass in kilodaltons. Molecular Plant 2011 4, 854-868DOI: (10.1093/mp/ssr025) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 10 OsRMR1 Did Not Interact with the Two Major Rice Storage Proteins Prolamin and Glutelin. Rice seed storage proteins glutelin, prolamin, globulin/albumin were isolated according to their different solubilities and were conjugated to column resins. The BY-2 culture media containing the overexpressed OsRMR1 NT-T7 protein was applied onto these columns. Both flow through (FT) and eluted proteins from the columns were subjected to SDS–PAGE and immunoblot analysis using anti-T7 antibodies. Recombinant OsRMR1 NT-T7 proteins were detected in FT fractions of each column and the elution fraction of the globulin/albumin column but not the prolamin and glutelin columns (indicated with asterisk). An extra band of ≈18 kDa with unknown identify was also detected in the eluted fraction from globulin/albumin column (lane 3, double asterisks). M, molecular mass in kilodaltons. Molecular Plant 2011 4, 854-868DOI: (10.1093/mp/ssr025) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 11 Working Model of Storage Protein Sorting in Rice Seeds. Certain rice storage proteins are synthesized in the ER and subsequently incorporated into vesicles to form PB I or ER body. Some other rice storage proteins are recognized by OsRMR1 at the Golgi apparatus or trans-Golgi network (TGN) and packed into the OsRMR1-positive organelles or storage PVCs (sPVCs) with electron-dense core for further delivery to PB II or PSV. ER, endoplasmic reticulum; TGN, trans-Golgi network; PB I, protein body type I; PB II, protein body type II; sPVC, storage prevacuolar compartment. Molecular Plant 2011 4, 854-868DOI: (10.1093/mp/ssr025) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions