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1 Supplemental information Activity of the purified plant ABC transporter NtPDR1 is stimulated by diterpenes and sesquiterpenes involved in constitutive and induced defenses Baptiste Pierman ǂ1, Frédéric Toussaint ǂ1, Aurélie Bertin, Daniel Lévy, Nicolas Smargiasso, Edwin De Pauw, Marc Boutry ǂ2 ǂ Institut des Sciences de la Vie, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium, the Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, 75005, Paris, France and Sorbonne Universités, UPMC Univ Paris 06, 75005, Paris, France, Mass Spectrometry Laboratory, Molecular Systems Research Unit, University of Liège, B-4000 Liège, Belgium 1 Both authors contributed equally to this work 2 To whom correspondence should be addressed (Tel: ; Fax: ; marc.boutry@uclouvain.be) Figure S1. Schematic representation of the construct used for NtPDR1 expression in N. tabacum BY-2 cells. Figure S2. Solubilization of 10His-StrepII-TEV-NtPDR1 by different detergents. Figure S3. Prediction of NtPDR1 glycosylation sites. Figure S4. Annotated spectrum of glycopeptide [WNHIVPGGNETLGSTVVK - GlcNAc2Man3 + HexNAc2Fuc1Xyl1]. Figure S5. Analysis of NtPDR1 reconstituted in asolectin liposomes. Figure S6. Analysis of NpPDR5 reconstituted in asolectin liposomes. S1

2 Figure S1 Figure S1. Schematic representation of the construct used for NtPDR1 expression in N. tabacum BY-2 cells. A. Amino acid sequence of the tag fused to the amino terminal end of NtPDR1. The 10 Histidine tag is represented in blue, the StrepII tag in green, the sequence recognized by the tobacco etch virus protease (TEV) in red, and the beginning of the NtPDR1 sequence in brown. The star indicates the proteolytic site. (B) Nucleotide sequence of the tag fused to the amino terminal end of NtPDR1. The two first codons of NtPDR1 are shown in bold. S2

3 Figure S2 Figure S2. Solubilization of 10His-StrepII-TEV-NtPDR1 by different detergents. A microsomal fraction (2.5 mg protein) was incubated in one ml of solubilization medium (see Experimental Procedures) supplemented with 1% of the indicated detergent. After incubation for 30 min at 4 C, the samples were centrifuged for 15 min at 100,000 g. The pellet (P) was resuspended in the same volume of solubilization buffer. Thirty µl of the supernatant (SN) and of the resuspended pellet (P) were analyzed by western blotting using HisProbe. C-: negative control without detergent. SDS: sodium dodecyl sulfate; DDM: n- dodecyl β-d-maltoside; MNG: decyl maltose neopentyl glycol; OG: octyl-β-glucoside; LDAO: lauryldimethyl amine oxide; CHAPS: 3-(3-cholamidopeopyl)-dimethylamino-2-hydroxy-1- propanesulfonate; C14E8: tetradecyl octaethylene glycol ether; TX-100: triton X-100 S3

4 Figure S3 Figure S3. Prediction of NtPDR1 glycosylation sites. NtPDR1 topology was modelled using the software Octopus (1) and drawn using Protter (2). Green and red boxes represent glycosylated and nonglycosylated NXS/T motifs, respectively. The number indicates the Asn residue. S4

5 Figure S4 Figure S4. Annotated spectrum of glycopeptide [WNHIVPGGNETLGSTVVK - GlcNAc2Man3 + HexNAc2Fuc1Xyl1]. Both glycan and peptide were found to be fragmented. P stands for complete peptide, i.e. fragments containing the complete sequence with an additional glycan moiety. Several fragment ions confirm the presence of fucose and xylose in the glycan moiety. S5

6 Figure S5 Figure S5. Analysis of NtPDR1 reconstituted in asolectin liposomes. A. NtPDR1-containing liposomes (5 µg proteins) were subjected to centrifugation through a discontinuous sucrose gradient as described in the Experimental Procedures section. Upon centrifugation, fractions of 300 µl were harvested and 30 µl of each fraction were analyzed by western blotting with antibodies against NtPDR1 (3). Reconstituted NTPDR1 (0.5 µg) was used as an input control. B. Reconstituted NtPDR1 (1.5 µg) was incubated in the presence or absence of 1 µg trypsin, 1.6 % (W/V) Triton X-100, and 20 µg trypsin inhibitor for 20 min at 37 C. Then, the samples were analyzed by western blotting with an antibody against NtPDR1. S6

7 Figure S6 Figure S6. Analysis of NpPDR5 reconstituted in asolectin liposomes. A. NpPDR5-containing liposomes (5 µg proteins) were centrifuged through a discontinuous sucrose gradient as described in the Experimental Procedures section. Upon centrifugation, fractions of 300 µl were harvested and 30 µl of each fraction were analyzed by western blotting and detected with antibodies against NpPDR5 (4). Reconstituted NpPDR5 (0.5 µg) was used as an input control. B. Reconstituted NpPDR5 (1.5 µg) was incubated in the presence or absence of 1 µg trypsin, 1.6% (W/V) Triton X-100, and 20 µg trypsin inhibitor for 20 min at 37 C. Then, the samples were analyzed by western blotting and detected by an antibody against NpPDR5. S7

8 References 1. Viklund, H., and Elofsson, A. (2008) OCTOPUS: Improving topology prediction by two-track ANN-based preference scores and an extended topological grammar. Bioinformatics 24, Omasits, U., Ahrens, C. H., Müller, S., and Wollscheid, B. (2014) Protter: interactive protein feature visualization and integration with experimental proteomic data. Bioinformatics 30, Crouzet, J., Roland, J., Peeters, E., Trombik, T., Ducos, E., Nader, J., and Boutry, M. (2013) NtPDR1, a plasma membrane ABC transporter from Nicotiana tabacum, is involved in diterpene transport. Plant Mol. Biol. 82, Bienert, M. D., Siegmund, S. E. G., Drozak, A., Trombik, T., Bultreys, A., Baldwin, I. T., and Boutry, M. (2012) A pleiotropic drug resistance transporter in Nicotiana tabacum is involved in defense against the herbivore Manduca sexta. Plant J. 72, S8