Features of the parkin/ariadne-like ubiquitin ligase, HHARI, that regulate its interaction with the ubiquitin-conjugating enzyme, Ubch7.
We recently reported the identification of a RING finger-containing protein, HHARI (human homologue of Drosophila ariadne), which binds to the human ubiquitin-conjugating enzyme UbcH7 in vitro. We now demonstrate that HHARI interacts and co-localizes with UbcH7 in mammalian cells, particularly in the perinuclear region. We have further defined a minimal interaction region of HHARI comprising residues 186-254, identified individual amino acid residues essential for the interaction, and determined that the distance between the RING1 finger and IBR (in between RING fingers) domains is critical to maintaining binding. We have also established that the RING1 finger of HHARI cannot be substituted for by the highly homologous RING finger domains of either of the ubiquitin-protein ligase components c-CBL or Parkin, despite their similarity in structure and their independent capabilities to bind UbcH7. Furthermore, mutation of the RING1 finger domain of HHARI from a RING-HC to a RING-H2 type abolishes interaction with UbcH7. These studies demonstrate that very subtle changes to the domains that regulate recognition between highly conserved components of the ubiquitin pathway can dramatically affect their ability to interact.
Molecular Medicine Unit and the Leeds Dental Institute, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds LS9 7TF, United Kingdom.
H C
HC
Ardley
N G
NG
Tan
S A
SA
Rose
A F
AF
Markham
P A
PA
Robinson
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D029721:Drosophila Proteins; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry*; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D016335:Zinc Fingers
C412166:Ari-1 protein, Drosophila; D002352:Carrier Proteins; D029721:Drosophila Proteins; D019476:Insect Proteins; D016271:Proto-Oncogene Proteins c-myc; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C419193:ARIH1 protein, human; D044767:Ubiquitin-Protein Ligases; D008025:Ligases