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Ian Donaldson

Analysis of Mammalian Peroxin Interactions Using a Non-transcription-based Bacterial Tw... - 0 views

shared by Ian Donaldson on 07 Jul 09 - Cached
  • Pex5p, interacts with its docking protein, Pex14p
    • Ian Donaldson
    • Ian Donaldson
    • Ian Donaldson
       
      PMID 12096124
      http://wodaklab.org/iRefWeb/compareInteractions/list?pubmed=12096124
      Both BIND and MINT report an interaction between PEX14 and PEX5.
      MINT: http://wodaklab.org/iRefWeb/interaction/show/85018
      BIND: http://wodaklab.org/iRefWeb/interaction/show/78001
      Both choose the same sequence for PEX14 but BIND cites splice isoform b of PEX5 (NP_000310.2) while MINT cites isoform d (P50542, NP_0011224498.1 and NP_0011224497.1). So the disagreement is due to splice isoform choice.
  • Using this approach, we were able to confirm that the C3HC4 domain of Pex12p, but not the full-length molecule, interacts with Pex5p
    • Ian Donaldson
       
      Both BIND and MINT report an interaction between PEX12 and PEX5
      BIND: http://wodaklab.org/iRefWeb/interaction/show/184407
      MINT: http://wodaklab.org/iRefWeb/interaction/show/212532
      Both choose the same sequence for PEX12 but BIND cites splice isoform b of PEX5 (NP_000310.2) while MINT cites isoform d (P50542, NP_0011224498.1 and NP_0011224497.1). So the disagreement is due to splice isoform choice.
  • For Pex5p, we mapped the site required for PTS1 and Pex12p interaction to the tetratricopeptide repeat domains labeled 2–8 and 2–9, respectively (Fig. 4).
  • ...6 more annotations...
  • For Pex5p, we mapped the site required for PTS1 and Pex12p interaction to the tetratricopeptide repeat domains labeled 2–8 and 2–9, respectively (Fig. 4).
  • These results, combined with the observation that Pex12p(275–359) interacts with Pex5p in the y-2HS (see Refs. 29 and 34, and see Fig. 4), suggest that Pex5p has distinct binding sites for the C3HC4 RING finger domain of Pex12p and PTS1 proteins and that Pex5p and Pex5p-Pex12p(275–359) have similar PTS1 binding properties
  • Several lines of evidence suggest that this complex is in fact a homodimer of Pex14p. (i) Pex14p can homodimerize when it is expressed in E. coli (Fig. 1); (ii) the cross-linked Pex14p-containing complex of CHO cells migrates during SDS-PAGE to a similar distance as the dimerized fraction of bacterially expressed (His)6-Pex14p (data not shown); and (iii) the cross-linked Pex14p-containing complex is not recognized by antisera raised against the Pex14p-interacting peroxins Pex5p, Pex13p, and Pex19p (data not shown). Interestingly, even in the absence of cross-linker a small but significant fraction of Pex14p migrated as a dimer on SDS-PAGE under non-reducing conditions (Fig. 3). However, deletion analysis studies revealed that the dimeric Pex14p structure is not maintained by disulfide bonding between the subunits but by its coiled-coil structure (see below).
    • Ian Donaldson
       
      Both BIND and MINT report that PEX14 exists as a homodimer
      http://wodaklab.org/iRefWeb/interaction/show/333798
  • Interestingly, this domain of Pex13p was also found to interact weakly with itself, at least in the y-2HS (Fig. 1).
    • Ian Donaldson
       
      Both BIND and HPRD note that PEX13 interacts with itself.
      http://wodaklab.org/iRefWeb/interaction/show/48384
      This was noted in the paper as a week interaction involving only a specific domain, perhaps explaining why MINT chose not to record this interaction.
  • However, in vitro reconstitution experiments of a putative peroxisomal protein import complex consisting of PTS1 ligand, Pex5p, Pex14p, and the SH3 domain of Pex13p (amino acids 236 to 403) demonstrated that Pex14p, but not the SH3 domain of Pex13p, interacts with Pex5p loaded with PTS1 cargo (Table II). In addition, these experiments show that Pex14p can bind simultaneously to Pex5p and Pex13p/SH3 (Table II). Summarized, these results suggest that in mammals (i) Pex14p, and not Pex13p, functions as the docking protein for cargo-loaded Pex5p, and (ii) the Pex5p-Pex13p interaction is bridged by Pex14p (Table II).
    • Ian Donaldson
       
      Both BIND and MINT note an interaction between PEX14 and PEX13.
      http://wodaklab.org/iRefWeb/interaction/show/109930
    • Ian Donaldson
       
      Both BIND and MINT report an interaction between PEX14 and PEX5.
      MINT: http://wodaklab.org/iRefWeb/interaction/show/85018
      BIND: http://wodaklab.org/iRefWeb/interaction/show/78001
      Both choose the same sequence for PEX14 but BIND cites splice isoform b of PEX5 (NP_000310.2) while MINT cites isoform d (P50542, NP_0011224498.1 and NP_0011224497.1). So the disagreement is due to splice isoform choice.
  • This validation study yielded 9 different interacting peroxin pairs; four pairs (Pex14p-Pex5p, Pex19p-Pex3p, Pex19p-Pex16p, and Pex19p-Pex11pß) were found to interact in both two-hybrid systems, three pairs (Pex19p-Pex10p, Pex19p-Pex12p, and Pex19p-Pex13p) were only detected in the y-2HS, and two pairs (Pex14p-Pex14p and Pex14p-Pex19p) interacted solely in the b-2HS.
    • Ian Donaldson
       
      PEX19 interactions:
      PEX19-PEX3: http://wodaklab.org/iRefWeb/interaction/show/259182
      PEX19-PEX11B: http://wodaklab.org/iRefWeb/interaction/show/210687
      PEX19-PEX12: http://wodaklab.org/iRefWeb/interaction/show/106817
      PEX19-PEX13: http://wodaklab.org/iRefWeb/interaction/show/139982
      PEX19-PEX16: http://wodaklab.org/iRefWeb/interaction/show/318750
      PEX19-PEX14: http://wodaklab.org/iRefWeb/interaction/show/270311
      In summary
      BIND and MINT both miss PEX19-PEX10
      BIND misses PEX10-PEX16
      MINT misses PEX19-PEX12 and PEX19-PEX13
      Neither BIND nor MINT appear to use the assay system(s) used to evidence the interaction as a consistant criteria for curation of the interaction.
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