PMC

The TPR domain of TWD1 domain is responsible for the interaction with AtMRP1. TWD1 fragments fused to an GAL4 binding domain tested for interaction with AD-MRP1 are represented by boxes as follows: PPIase, cis-trans-peptidyl prolyl isomerase domain; TPR, tetratrico peptide repeat; C, calmodulin-binding domain; M, membrane anchor. Amino acid positions are indicated. Negative controls are from top to bottom: BD-BusB/AD vector, BD vector/AD-MRP1, and AD vector/BD vector. Activation of histidine growth reporter (growth on -HIS) is indicated by + and - and LacZ reporter activities are displayed as mean units per mg; error bars represent standard deviations from three to five independent transformants.

TWD1 binds to and modulates uptake of ABC transporter model substrates into vacuoles. (A) Uptake of ¹⁴C-metolachlor-GS and ³H-estradiol-β-glucuronide were carried out in the absence (control) or presence of each 1 μM purified TWD1-3 protein (+TWD1-3) and 1 μM calmodulin (+CaM), respectively. Conjugate uptake was measured with five replicas for each time point. Relative activities are displayed as means with standard deviations from three independent vacuole preparations. Import activities being statistically different (Mann-Whitney U test, p > 0.05) compared with control experiments are indicated by an asterisks. (B) Purified TWD1-3 protein binds specifically to vacuolar membranes. Arabidopsis vacuoles (+vac) were incubated with purified TWD1-3 or 14-3-3 protein (14-3-3-GF14-ω; ) and pelleted by ultracentrifugation. Equal volumes of pellets (P) and supernatants (SN) were separated by PAGE and TWD1-3, and 14-3-3-GF14-ω were detected using anti-pentaHIS antibodies.

The PAS1 but not ROF1 TPR domain of TWD1 interacts with AtMRP1 in yeast two-hybrid assays. (A) Domain structures of high-molecular-weight FKBPs from Arabidopsis. Functional domains of At-FKBP12, TWD1, ROF1, ROF2, and PAS1 are boxed and indicated as follows: cis-trans peptidy-prolyl isomerase domain (FKBP), red; tetratrico peptide repeats (TPR) blue; CaMbd, violet; membrane anchor, green. (B) Specificity of PAS1-AtMRP1 interaction in the yeast two-hybrid system. TPR fragments of PAS1 and ROF1 were fused to a GAL4 binding domain and tested for interaction with the C-terminus of AtMRP1 including the CaMbd (AD-MRP1+CaMbd) or lacking the CaMbd (AD-MRP1-CaMbd). Negative controls are from top to bottom: AD-MRP1/BD vector and AD vector/BD vector. Activation of histidine growth reporter (growth on -HIS) is indicated by + and - and LacZ reporter activities are displayed as mean units per mg; error bars represent standard deviations from three to five independent transformants.

A calmodulin-binding domain in AtMRP1 mediates interaction to TWD1. (A) Alignment of a putative calmodulin-binding domain (CaMbd) of AtMRP1 (MIPS code At1g30400) and AtMRP2 (At2g34660) with selected CaMbds. CaMbds of different origin were taken from Geisler et al. () and aligned using MegAlign (DNAStar, Madison, WI). Position of conserved residues essential for calmodulin binding are boxed, and conserved residues are printed in bold. (B) Calmodulin overlay the AtMRP1 C-terminus. Soluble E. coli extracts of cells expressing the MRP1 C-terminus (AtMRP1-1: residues 1217-1392) were transferred onto nitrocellulose and probed using anti-RGSHis antisera or biotinylated calmodulin (CaM-overlay) in the presence (+Ca) or absence of calcium (-Ca). The asterisk marks binding of E. coli proteins that were found also with vector control lysates. (C) The putative CaMbd domain of AtMRP1 is mainly responsible for the interaction with TWD1. AD-MRP1 fragment with (▪) and without putative CaMbd (□) are tested against indicated TWD1 fragments fused to a GAL4 binding domain. Activation of histidine growth reporter (growth on -HIS) is indicated by + and - and LacZ reporter activities are displayed as mean units per mg; error bars represent standard deviations from three to five independent transformants. (D) Calmodulin does not affect the interaction between TWD1 and AtMRP1 in vitro. A TWD1 affinity matrix was incubated with cleared E. coli lysates containing the expressed C-termini of AtMRP1 (control, lane 1). Some reactions were preincubated with spinach calmodulin in the presence (+Ca, lane 2) or absence of calcium (-Ca, lane 3). Matrix-eluted proteins were separated by PAGE and immunoprobed against anti-pentaHIS.

TWD1 interacts specifically with the C-termini of AtMRP1 and AtMRP2. (A) Yeast two-hybrid analysis of TWD1 interacting clones. Two-hybrid screening of an Arabidopsis cDNA library using TWD1 (BD-BusB) resulted in the identification of AtMRP1 (clone BE11). Homologous stretches of selected AtMRPs were fused to a GAL4 activation domain and tested for interaction with the soluble part of TWD1 (BD-BusB). Negative controls are from top to bottom: BD-BusB/AD vector, BD vector/AD-MRP1, and AD vector/BD vector. Activation of histidine growth reporter (growth on -HIS) is indicated by + and -; LacZ reporter activities are displayed as units per mg; error bars represent standard deviations from three to five independent transformants. (B) Arabidopsis MRP-like ABC transporters cluster into two clades. The tree was modified from Kolukisaoglu et al. () and identity and accession numbers can be deduced from there. Positive and negative two-hybrid interactions of AtMRPs tested against TWD1 in A are indicated with + and -, respectively. (C) In vitro interaction between TWD1 and AtMRP1. A TWD1 affinity matrix was incubated with cleared E. coli lysates containing the expressed C-termini of AtMRP1 (lane 1) or the vector control (lane 3). As negative control, empty Affigel beads were incubated with the C-terminus of AtMRP1-1 lysate (lane 2). Matrix-eluted proteins were separated by PAGE and immunoprobed against penta-His and anti-RGSHis recognizing the TWD1-3 and AtMRP1-1 peptides, respectively. Note that unproportional staining of TWD1-3 and AtMRP1-1 peptides is due to different affinities of the used antisera toward their antigens (see text).

In vivo interaction between TWD1 and AtMRP1. (A) TWD1-HA overlaps with vacuolar and plasma membrane fractions in a continuous sucrose gradient. Arabidopsis microsomal fractions from plants expressing TWD1-HA were separated by linear sucrose gradient centrifugation and equal amounts of protein of selected fractions were probed with anti-HA and anti-MRP1. Origin of immunopositive fractions was ascertained by Western blotting using antisera against the marker proteins vacuolar PPase, ER localized BIP and the plasma membrane-bound P-type H⁺-ATPase (). (B) Immunoprecipitation of a TWD1-AtMRP1 complex. Microsomal membranes from Arabidopsis wild-type (lanes 1-5) or TWD-HA plants (lanes 6-9) were cross-linked with DTBP, solubilized using 0.1% Brij 35 and 0.05% CHAPS and immunoprecipitated using an anti-AtMRP1 or anti-HA affinity matrix, respectively. Immunoprecipitated proteins were separated by 7.5% (lanes 1, 2, and 5), 12.5% (lanes 3, 4, 6, and 7) or 4-12% PAGE (lanes 8 and 9) in the presence (+) or absence of DTT (-) and probed with anti-AtMRP1 (lanes 1-2, 8-9), anti-TWD1 (lanes 3-5), and anti-HA (lanes 6-9), respectively. As negative controls, unspecific binding of proteins to empty protein G was monitored (lanes 2 and 4). Note that the size difference of the coprecipitated wild-type TWD1 in lane 3 having a slightly smaller weight than the HA-epitope-tagged TWD1 (lane 6) is due to the lack of the HA-epitope. Molecular size markers on the left and right correspond to lanes 1-2, and 5 and lanes 3-4, 6-7, respectively; positions of TWD1 and AtMRP1 are indicated.

AtMRP1 is localized on the central vacuolar membrane. (A) Structural model of AtMRP1. Functional domains of AtMRP1 are boxed. A putative calmodulin-binding domain (CaMbd) and extension of the isolated two-hybrid clone BE11 as well as the sites of insertion of enhanced GFPs (EGFP) into constructs pGPTV-MRP1-12 and pGPTV-MRP1-13 are indicated. Abbreviations are as follows: NTE, N-terminal extension; TMD, transmembrane domain; NBF, nucleotide binding fold; LR, linker region; CTE, C-terminal extension. (B) Laser scanning confocal analysis of plant material expressing a GFP-tagged version of AtMRP1. Images represent internal optical sections generated by CLSM from root material of heterozygous plants expressing ectopically (i and ii) and onion epidermis cells transiently expressing AtMRP1-EGFP (iv and v). The tonoplast surrounding the cytoplasma including the nucleus is indicated by asterisks; small GFP-labeled cytoplasmic structures obtained with construct pGPTV-MRP1-13 are marked by arrows. (i) Root apex. (ii) Close-up of the root hair zone. (iii) Onion epidermis cell transiently expressing the tonoplast marker KCO1-GFP (; red color). (iv) Onion epidermis cell expressing AtMRP1-EGFP (construct pGPTV-MRP1-12, green color). (v) Onion epidermis cell expressing AtMRP1-EGFP (construct pGPTVMRP1-13, green color). (vi) Same cells in a bright field. (C) Arabidopsis microsomal fractions from wild-type plants separated by linear sucrose gradient centrifugation were probed with anti-AtMRP1 antisera. Origin of immunopositive fractions was ascertained by Western blots using antisera against the marker proteins vacuolar V-type H⁺-ATPase, ER localized BIP, and the plasma membrane-bound P-type H⁺-ATPase ().