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Open Access Research article

A genome-wide study of PDZ-domain interactions in C. elegans reveals a high frequency of non-canonical binding

Nicolas Lenfant1, Jolanta Polanowska13, Sophie Bamps12, Shizue Omi13, Jean-Paul Borg1 and Jérôme Reboul13*

Author Affiliations

1 Inserm, U891, CRCM, Marseille, F-13009, France; Institut Paoli-Calmettes, Marseille, F-13009, France; Univ Méditerranée, F-13007, Marseille, France

2 Institute of Integrative and Comparative Biology, Faculty of Biological Sciences, The University of Leeds, Leeds, LS2 9JT, UK

3 CIML, Parc Scientifique et Technologique de Luminy Case 906 - 13288 Marseille - Cedex 9, France

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BMC Genomics 2010, 11:671  doi:10.1186/1471-2164-11-671

Published: 26 November 2010

Additional files

Additional file 1:

Definition of consensus classes. Additional file 1 is a table describing the consensus classes used in this study. We defined for this study three extended consensus classes encompassing the different definitions available so far, so as to have the broadest definition of classes [1,10].

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Additional file 2:

PDZ domains cloning and annotations. Additional file 2 contains two tables (S1 and S2) listing for each PDZ domain identified, respectively the primer sequences and the Gene Ontology annotations. Supplemental Table S1: List of primers used to clone PDZ domains. Proteins names and IDs are given according to Wormbase WS150 [44]. For multiple PDZ domains in the same protein, a ".n" extension was added to the ID of the PDZ containing protein. This extension was numbered from the ATG (eg: F54E7.3.1 is the ID for the first PDZ domain of F54E7.3). When only one PDZ was present, protein ID was kept as such. Coordinates on PDZ domain containing proteins correspond to the splice form specified in the third column (PDZ domain containing protein ID). Each primer contains the B1 and B2 Gateway recombination cloning tail. Supplemental Table S2: Gene Ontology annotation based on experimental data for PDZ domains proteins. Gene ontology annotations were retrieved from Wormbase WS190 [16]. Experimental Evidence Codes: EXP: Inferred from Experiment, IDA: Inferred from Direct Assay, IPI: Inferred from Physical Interaction, IMP: Inferred from Mutant Phenotype, IGI: Inferred from Genetic Interaction, IEP: Inferred from Expression Pattern.

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Additional file 3:

PDZome network. Additional file 3 contains four tables (S3 to S6) listing the PDZome network interacting pairs and giving statistical analysis of the interactions. Supplemental Table S3: Two hybrid screen results. Gene names and ID are given according to Wormbase WS150[44]. Number of hits refers to the number of independent colonies identified and phenotypically tested for each interacting partner. Number of splice-forms identified or predicted in Wormbase WS150 and last 6 amino acids of each splice-form are given in cases were several splice-forms are identified or predicted. When sequencing from the N-terminus did not span the entire fragment, and thus the C-terminus was not experimentally confirmed, if any of the predicted splice-form had a C-terminal consensus motif, to be conservative, a consensus class was attributed. Consensus class type: [ST]X[YFWCMVILA] = 1; [YFWCMVILA]X[YFWCMVILA] = 2; [DE]X[YFWCMVILA] = 3 (X: any amino acid). Supplemental Table S4: Promiscuity and specificity of PDZ interactome network. Number of independent interacting proteins per PDZ domain, and number of PDZ domains interacting with each protein are given for AD-wrmcDNA and AD-ORFeome libraries two-hybrid screens. Supplemental Table S5: Promiscuity and specificity of PDZ interactome network: mean and median for number of interacting proteins per PDZ or vice versa. Supplemental Table S6: Number of interacting proteins per consensus class in network. Single hits: interacting proteins for which only one clone was identified in two-hybrid screens. Multiple hits: interacting proteins for which more than one clone was identified in two-hybrid screens.

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Additional file 4:

Functional annotation of interacting proteins. Additional file 4 contains three tables (S7 to S9) listing the functional annotations of the PDZ domain interacting proteins. Supplemental Table S7: Concise description, GO terms and KOG (EuKaryotic Orthologous Groups), for each interacting protein when available, retrieved from Wormbase WS 190 [16]; note that most attributes are inferred from electronic annotation. Experimental Evidence Codes: EXP: Inferred from Experiment, IDA: Inferred from Direct Assay, IPI: Inferred from Physical Interaction, IMP: Inferred from Mutant Phenotype, IGI: Inferred from Genetic Interaction, IEP: Inferred from Expression Pattern. Computational Analysis Evidence Codes: ISS: Inferred from Sequence or Structural Similarity, ISO: Inferred from Sequence Orthology, ISA: Inferred from Sequence Alignment, ISM: Inferred from Sequence Model, IGC: Inferred from Genomic Context, RCA: inferred from Reviewed Computational Analysis. Author Statement Evidence Codes: TAS: Traceable Author Statement, NAS: Non-traceable Author Statement. Curator Statement Evidence Codes: IC: Inferred by Curator, ND: No biological Data available. Automatically-assigned Evidence Codes: IEA: Inferred from Electronic Annotation. Supplemental Table S8: Classification of interacting proteins according to Cellular Components terms: integral to membrane, nucleus and other&unknown. Supplemental Table S9: Classification of interacting proteins according to Biological Processes: manual curation of annotations retrieved from Wormbase WS190 were used to define the 11 groups of processes shown in Figure 2.

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Additional file 5:

Verification of two-hybrid interacting pairs by co-immunoprecipitation. Additional file 5 contains four tables (S10 to S13) listing interacting proteins pairs tested and results. Supplemental Table S10 lists tested pairs involving PDZ domains and their respective two-hybrid identified interacting proteins possessing a free C-terminus. Supplemental Table S11 lists tested pairs involving PDZ domains and their respective two-hybrid identified interacting proteins using a B2 tailed construct. Supplemental Table S12 lists the 59 protein fragments that did not possess a C-terminal binding motif tested by co-immunoprecipitation in a C-terminally truncated form against their respective two hybrid interacting PDZs. Sequences where there was creation of a new C-terminal consensus sites after truncation are shown. Supplemental Table S13 lists the primers pairs used to amplify and clone truncated interacting protein fragments used in the co-immunoprecipitation experiment.

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Additional file 6:

Co-IP verification of yeast two hybrid interacting pairs identified in AD-wrmcDNA and AD-ORFeome screens using a B2 tailed construct. Additional file 6 is a figure showing the Co-IP verification of yeast two hybrid interacting pairs identified in AD-wrmcDNA and AD-ORFeome screens using a B2 tailed construct. (A) Schematic representation of PDZ domains carrying N-terminal 3XHA epitope tag and of their interacting protein, ending with the B2 tail, carrying N-terminal MYC epitope. (B) Each pair of constructs to be tested was co-expressed in 293T cells and co-IP was performed using cellular lysates subjected to precipitation with anti-HA sepharose. Presence of interacting protein upon precipitation was revealed by western blotting using anti-MYC serum. For each IP performed three panels are presented. Upper panel: IP reaction probed upon resolution on SDS-PAGE and blotting with anti-HA antibody detecting HA-PDZ domain; Middle panel: the same IP reaction probed with anti-MYC serum detecting ORF (MYC:ORF); lower panel: detection of expression of each ORF by probing total crude cellular extracts (input) with anti-MYC serum. Table summarizes the interaction pairs tested and color code is used to indicate the outcome (purple: interaction tested positive, grey: no interaction and yellow: inconclusive as one or both partners are not expressed). (C) Each ORF used in above co-IP experiment was also subjected to co-transfection and co-immunoprecipitation with empty pDEST-CMV-3xHA vector to serve as a negative control for the binding assay. Detection and analysis were performed as above.

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Additional file 7:

Negative controls of immunoprecipitations shown in Figure 4. Additional file 7 is a figure showing the test for unspecific binding of non-consensus C-terminally truncated proteins (MYC:MIRdCter) to irrelevant HA epitoped peptide in co-immunoprecipitation reaction corresponding to negative control of the experiment described in Figure 4. Each MYC:MIRdCter construct was co-expressed in 293T cells together with empty pDEST-CMV-3xHA vector and co-IPed using anti-HA sepharose beads. Binding of given protein upon precipitation was revealed by western blotting using anti-MYC serum. For each IP performed three panels are presented. Upper panel: IP reaction probed after resolution on SDS-PAGE and blotting with anti-HA antibody. Middle panel: the same IP reaction probed with anti-MYC serum detecting the truncated protein fragments (MYC:MIRdCter). Lower panel: detection of expression of each truncated protein fragment by probing total crude cellular extracts (input) with anti-MYC serum.

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