Screening for protein-DNA interactions by automatable DNA-protein interaction ELISA.

DNA-binding proteins (DBPs), such as transcription factors, constitute about 10% of the protein-coding genes in eukaryotic genomes and play pivotal roles in the regulation of chromatin structure and gene expression by binding to short stretches of DNA. Despite their number and importance, only for a minor portion of DBPs the binding sequence had been disclosed. Methods that allow the de novo identification of DNA-binding motifs of known DBPs, such as protein binding microarray technology or SELEX, are not yet suited for high-throughput and automation. To close this gap, we report an automatable DNA-protein-interaction (DPI)-ELISA screen of an optimized double-stranded DNA (dsDNA) probe library that allows the high-throughput identification of hexanucleotide DNA-binding motifs. In contrast to other methods, this DPI-ELISA screen can be performed manually or with standard laboratory automation. Furthermore, output evaluation does not require extensive computational analysis to derive a binding consensus. We could show that the DPI-ELISA screen disclosed the full spectrum of binding preferences for a given DBP. As an example, AtWRKY11 was used to demonstrate that the automated DPI-ELISA screen revealed the entire range of in vitro binding preferences. In addition, protein extracts of AtbZIP63 and the DNA-binding domain of AtWRKY33 were analyzed, which led to a refinement of their known DNA-binding consensi. Finally, we performed a DPI-ELISA screen to disclose the DNA-binding consensus of a yet uncharacterized putative DBP, AtTIFY1. A palindromic TGATCA-consensus was uncovered and we could show that the GATC-core is compulsory for AtTIFY1 binding. This specific interaction between AtTIFY1 and its DNA-binding motif was confirmed by in vivo plant one-hybrid assays in protoplasts. Thus, the value and applicability of the DPI-ELISA screen for de novo binding site identification of DBPs, also under automatized conditions, is a promising approach for a deeper understanding of gene regulation in any organism of choice.

An update on the ABCC transporter family in plants: many genes, many proteins, but how many functions?

The ABCC subfamily of the ATP binding cassette (ABC) transporters, which were formerly known as multidrug resistance-related proteins (MRPs), consists of closely related members found in all eukaryotic organisms. Although more than a decade of intensive research has elapsed since the first MRP protein was functionally characterised in Arabidopsis thaliana, knowledge of this particular transporter family is still limited in plants. Although ABCC proteins were originally defined as vacuolar pumps of glutathione-S (GS) conjugates, evidence, as well as speculation, on their endogenous functions inside the cell ranges from detoxification and heavy metal sequestration, to chlorophyll catabolite transport and ion channel regulation. The characterisation of knockout mutants in Arabidopsis has been pivotal for elucidation of different roles of ABCC transporters. However, a functional annotation for the majority of these transport proteins is still lacking, even in this model plant. On the one hand, this problem seems to be caused by functional redundancy between family members, which might lead to physiological complementation by a highly homologous gene in the mutant lines. On the other hand, there is growing evidence that the functional diversity of ABCC genes in Arabidopsis and other plants is far greater than previously assumed. For example, analysis of microarray expression data supports involvement of ABCC transporters in the response to biotic stress: particular changes in ABCC transcript levels are found, which are pathogen-specific and evoke distinct signalling cascades. Current knowledge about plant ABCC transporters indicates that novel and unexpected functions and substrates of these proteins are still waiting to be elucidated.

Comparative mutant analysis of Arabidopsis ABCC-type ABC transporters: AtMRP2 contributes to detoxification, vacuolar organic anion transport and chlorophyll degradation.

The enormous metabolic plasticity of plants allows detoxification of many harmful compounds that are generated during biosynthetic processes or are present as biotic or abiotic toxins in their environment. Derivatives of toxic compounds such as glutathione conjugates are moved into the central vacuole via ATP-binding cassette (ABC)-type transporters of the multidrug resistance-associated protein (MRP) subfamily. The Arabidopsis genome contains 15 AtMRP isogenes, four of which (AtMRP1, 2, 11 and 12) cluster together in one of two major phylogenetic clades. We isolated T-DNA knockout alleles in all four highly homologous AtMRP genes of this clade and subjected them to physiological analysis to assess the function of each AtMRP of this group. None of the single atmrp mutants displayed visible phenotypes under control conditions. In spite of the fact that AtMRP1 and AtMRP2 had been described as efficient ATP-dependent organic anion transporters in heterologous expression experiments, the contribution of three of the AtMRP genes (1, 11 and 12) to detoxification is marginal. Only knockouts in AtMRP2 exhibited a reduced sensitivity towards 1-chloro-2,4-dinitrobenzene, but not towards other herbicides. AtMRP2 but not AtMRP1, 11 and 12 is involved in chlorophyll degradation since ethylene-treated rosettes of atmrp2 showed reduced senescence, and AtMRP2 expression is induced during senescence. This suggests that AtMRP2 is involved in vacuolar transport of chlorophyll catabolites. Vacuolar uptake studies demonstrated that transport of typical MRP substrates was reduced in atmrp2. We conclude that within clade I, only AtMRP2 contributes significantly to overall organic anion pump activity in vivo.

Genomics of plant ABC transporters: the alphabet of photosynthetic life forms or just holes in membranes?

The large number of ABC transporters in the Arabidopsis genome was made responsible for known as well as unexpected aspects in plant development. The combination of classical transport experiments with functional genomics approaches helped unravel some of these effects. Yet questions concerning the importance of this large number of ABC transporters in eukaryotic photosynthesizing organisms remain open. Phylogenomic analyses of whole genome sequence data reveal comparable sizes and composition between algae and higher plants within this protein family. Although this indicates the significance of ABC transporters in plants, several other questions remain to be answered.

Disruption of AtMRP4, a guard cell plasma membrane ABCC-type ABC transporter, leads to deregulation of stomatal opening and increased drought susceptibility.

ATP-binding cassette (ABC) transporters are membrane proteins responsible for cellular detoxification processes in plants and animals. Recent evidence shows that this class of transporters may also be involved in many other cellular processes. Because of their homology with human multidrug resistance-associated proteins (MRP), cystic fibrosis transmembrane conductance regulator (CFTR) and sulfonylurea receptor (SUR), some plant ABC transporters have been implicated in the regulation of ion channel activities. This paper describes an investigation of the AtMRP4 gene and its role in stomatal regulation. Reporter gene studies showed that AtMRP4 is highly expressed in stomata and that the protein is localized to the plasma membrane. Stomatal aperture in three independent atmrp4 mutant alleles was larger than in wild-type plants, both in the light and in the dark, resulting in increased water loss but no change in the photosynthetic rate. In baker's yeast, AtMRP4 shows ATP-dependent, vanadate-sensitive transport of methotrexate (MTX), an antifolate and a substrate of mammalian MRPs. Treatment with MTX reduced stomatal opening in wild-type plants, but had no effect in atmrp4 mutants. These results indicate the involvement of AtMRP4 in the complex regulation of stomatal aperture.

TWISTED DWARF1, a unique plasma membrane-anchored immunophilin-like protein, interacts with Arabidopsis multidrug resistance-like transporters AtPGP1 and AtPGP19.

Null-mutations of the Arabidopsis FKBP-like immunophilin TWISTED DWARF1 (TWD1) gene cause a pleiotropic phenotype characterized by reduction of cell elongation and disorientated growth of all plant organs. Heterologously expressed TWD1 does not exhibit cis-trans-peptidylprolyl isomerase (PPIase) activity and does not complement yeast FKBP12 mutants, suggesting that TWD1 acts indirectly via protein-protein interaction. Yeast two-hybrid protein interaction screens with TWD1 identified cDNA sequences that encode the C-terminal domain of Arabidopsis multidrug-resistance-like ABC transporter AtPGP1. This interaction was verified in vitro. Mapping of protein interaction domains shows that AtPGP1 surprisingly binds to the N-terminus of TWD1 harboring the cis-trans peptidyl-prolyl isomerase-like domain and not to the tetratrico-peptide repeat domain, which has been shown to mediate protein-protein interaction. Unlike all other FKBPs, TWD1 is shown to be an integral membrane protein that colocalizes with its interacting partner AtPGP1 on the plasma membrane. TWD1 also interacts with AtPGP19 (AtMDR1), the closest homologue of AtPGP1. The single gene mutation twd1-1 and double atpgp1-1/atpgp19-1 (atmdr1-1) mutants exhibit similar phenotypes including epinastic growth, reduced inflorescence size, and reduced polar auxin transport, suggesting that a functional TWD1-AtPGP1/AtPGP19 complex is required for proper plant development.

Family business: the multidrug-resistance related protein (MRP) ABC transporter genes in Arabidopsis thaliana.

Despite the completion of the sequencing of the entire genome of Arabidopsis thaliana (L.) Heynh., the exact determination of each single gene and its function remains an open question. This is especially true for multigene families. An approach that combines analysis of genomic structure, expression data and functional genomics to ascertain the role of the members of the multidrug-resistance-related protein ( MRP) gene family, a subfamily of the ATP-binding cassette (ABC) transporters from Arabidopsis is presented. We used cDNA sequencing and alignment-based re-annotation of genomic sequences to define the exact genic structure of all known AtMRP genes. Analysis of promoter regions suggested different induction conditions even for closely related genes. Expression analysis for the entire gene family confirmed these assumptions. Phylogenetic analysis and determination of segmental duplication in the regions of AtMRP genes revealed that the evolution of the extraordinarily high number of ABC transporter genes in plants cannot solely be explained by polyploidisation during the evolution of the Arabidopsis genome. Interestingly MRP genes from Oryza sativa L. (rice; OsMRP) show very similar genomic structures to those from Arabidopsis. Screening of large populations of T-DNA-mutagenised lines of A. thaliana resulted in the isolation of AtMRP insertion mutants. This work opens the way for the defined analysis of a multigene family of important membrane transporters whose broad variety of functions expands their traditional role as cellular detoxifiers.