Volume 5, Issue 1, Pages 43-62 (January 2012) The Potato Sucrose Transporter StSUT1 Interacts with a DRM-Associated Protein Disulfide Isomerase  Krügel Undine , He Hong-Xia , Gier Konstanze , Reins Jana , Chincinska Izabela , Grimm Bernhard , Schulze Waltraud X. , Kühn Christina   Molecular Plant  Volume 5, Issue 1, Pages 43-62 (January 2012) DOI: 10.1093/mp/ssr048 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Summary of mass spectrometric analyses. (A) Functional classification of detergent-resistant membrane proteins from potato source leaves identified by LC–MS/MS. A total of 92 different proteins have been identified. The complete set of proteins is given in Table 1. (B) Functional classification of co-immunoprecipitated proteins interacting with StSUT1 identified by LC–MS/MS analysis. Identification of proteins is based on the typical spectrum of single peptides and peptide sequences. The result of one representative experiment is given. A total of 47 SUT1-interacting proteins have been identified and the complete set of proteins is given in Table 2. Putative functions were determined by database comparison (UniProt, www.expasy.org/uniprot; NCBI, www.ncbi.nlm.nih.gov/entrez). Molecular Plant 2012 5, 43-62DOI: (10.1093/mp/ssr048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Protein-protein interactions in yeast by the split ubiquitin system. (A) Growth of the yeast strain L40ccU transformed with the SlSUT2 cDNA in the bait-vector Cub and the PDI cDNA in the prey vector Nub on selective medium (without His, Leu, Trp, and in the presence of 40 mM 3-AT). (B) The yeast strain L40ccU shows lacZ activity in a qualitative β-galactosidase activity assay if co-transformed with Cub bait vectors expressing sucrose transporter StSUT1, SlSUT2, or StSUT4, and the PDI cDNA in the Nub prey plasmid indicative for protein–protein interaction. Molecular Plant 2012 5, 43-62DOI: (10.1093/mp/ssr048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Characterization of the SUT-interacting PDI. (A) Schematic model of PDI structure. The protein disulfide isomerase interacting with all three known sucrose transporters from potato contains two highly conserved thioredoxin domains, Trx1 and Trx2, and a conserved ERp29 domain from the PDI-like chaperone protein ERp29. (B–D) Sub-cellular localization of the protein disulfide isomerase (PDI). The PDI fused to YFP is detectable in the ER (B), in vesicles (C) and at the plasma membrane in microdomain-like compartments (D). (C–D) Maximum projections, B single scans. YFP fluorescence was detected after excitation with 488 nm; mCherry fluorescence was detected after excitation with 543 nm. Molecular Plant 2012 5, 43-62DOI: (10.1093/mp/ssr048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Confirmation of Protein–Protein Interactions by Bimolecular Fluorescence Complementation (BiFC). Bait and prey proteins were fused via GATEWAY-based cloning into pE-SPYNE and pE-SPYCE. Leaves from N. benthamiana have been infiltrated with Agrobacterium-suspension transformed with Split YFP vectors and confocal analysis of YFP fluorescence in the leaf epidermis was performed 3–4 d after co-infiltration. (A) StSUT1 in SPYNE co-infiltrated with PDI in SPYCE. (B) StSUT4 in SPYNE co-infiltrated with PDI in SPYCE. (C) StSUT4 in SPYCE with PDI in SPYCE used as a negative control (both cDNA cloned in the same vector). (D) StSUT4 in SPYNE co-infiltrated with StSUT1 in SPYCE served as positive control. (E) AtTPK4 in SPYNE co-infiltrated with PDI in SPYCE used as negative control. (F) PDI in SPYCE with PDI in SPYNE shows the ability of the PDI to form homodimers. In (A, B, D, F), epidermal cells show YFP fluorescence in the ER, whereas, in (C, E), no or only weak fluorescence is detectable, which is not associated with cellular structures. Chlorophyll autofluorescence is shown in red; YFP fluorescence is shown in green for better contrast. Molecular Plant 2012 5, 43-62DOI: (10.1093/mp/ssr048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 Immunodetection of sucrose transporters by Western Blot Analysis. (A) SDS–PAGE of the potato microsomal fraction (MF), endomembrane fraction (EF), and plasma membranes (PM) after two-phase partitioning. Immunodetection was performed with StSUT1-specific antibodies. StSUT1 is detectable not only in the plasma membrane, but also in endomembranes. (B) Western blot analysis of the dimerization behavior of StSUT1 in the presence or absence of the PDI. Yeast cells of the strain SuSy7 ura3 (1) were transformed with either the empty vector pDR195 (2), or co-transformed with StSUT1 in pDR195 and the empty Nub vector (3), or with StSUT1 in pDR195 and PDI in Nub (4). Microsomal membranes have been isolated without addition of DTT and separated on SDS–PAGE. Equal amounts of protein are loaded. Immunodetection was performed using StSUT1-specific antibodies. Dimerization of the StSUT1 protein is not prevented in the presence of PDI. (C) Confirmation of StSUT4–PDI interaction by glutathione S-transferase (GST)-pull-down assay. The PDI was overexpressed in E. coli Rosetta™ (DE3) and GST protein alone was used as a negative control. StSUT4 was in vitro-transcribed and in vitro-translated with lysate of rabbit reticulocytes. Incorporation of radiolabeled [35S]-methionine allows detection via autoradiography. Lanes 1–3, GST; lanes 4–6, PDI-GST fusion; Lanes 1 + 4, supernatant after incubation of the TNT® reaction with conjugated MagneGST™ beads. Lanes 2 + 5, wash fraction; Lanes 3 + 6, elution fraction. Radiolabeled StSUT4 protein is detectable in the elution fraction only if PDI is bound to the GST beads. Molecular Plant 2012 5, 43-62DOI: (10.1093/mp/ssr048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 Real-Time PCR Analysis of PDI (A) and StSUT1 (B) Transcript Accumulation in a Light/Dark Cycle. Relative quantification was performed with ubiquitin primers as internal standard. The standard deviation (StDev) is given (two biological samples were analyzed with three technical replications). Student's t-test revealed significant differences between light and dark samples (with p < 0.01). Molecular Plant 2012 5, 43-62DOI: (10.1093/mp/ssr048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 7 Real-Time PCR Analysis of PDI Transcript Accumulation in Potato Source Leaves (A, C) or hydroponically grown entire plantlets (B, D). (A) Potato source leaves were kept in 2.5 mM EDTA supplemented with 20 mM β-mercaptoethanol or 5 μg ml−1 tunicamycin for 2.5 and 5 h. (B) Entire potato plantlets were hydroponically grown in medium supplemented with 10 mM DTT. (C) Potato source leaves were kept in 2.5 mM EDTA supplemented with 500 mM sucrose or 1 M glucose for 1.5 and 3 h. (D) Entire potato plantlets were hydroponically grown in medium supplemented with 500 mM sucrose or sorbitol for 2.5 and 5 h. Relative quantification was performed with ubiquitin primers as internal standard; the appropriate water control was subtracted. The StDev is given and a Student's t-test was performed (+ indicates samples differing significantly from time 0 h with p < 0.05). Molecular Plant 2012 5, 43-62DOI: (10.1093/mp/ssr048) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions