The cellular protein level of parkin is regulated by its ubiquitin-like domain.
Parkin is a ubiquitin-protein isopeptide ligase (E3) involved in ubiquitin/proteasome-mediated protein degradation. Mutations in the parkin gene cause a loss-of-function and/or alter protein levels of parkin. As a result, the toxic build-up of parkin substrates is thought to lead to autosomal recessive juvenile Parkinsonism. To identify a role for the ubiquitin-like domain (ULD) of parkin, we created a number of hemagglutinin (HA)-tagged parkin constructs using mutational and structural information. Western blotting and immunocytochemistry showed a much stronger expression level for HA-parkin residues 77-465 (without ULD) than HA-parkin full-length (with ULD). The deletion of ULD in Drosophila parkin also caused a sharp increase in expression of the truncated form, suggesting that the function of the ULD of parkin is conserved across species. By progressive deletion analysis of parkin ULD, we found that residues 1-6 of human parkin play a crucial role in controlling the expression levels of this gene. HA-parkin residues 77-465 showed ubiquitination in vivo, demonstrating that the ULD is not critical for parkin auto-ubiquitination; ubiquitination seemed to cluster on the central domain of parkin (residues 77-313). These effects were specific for the ULD of parkin and not transfection-, toxic-, epitope tag-, and/or vector-dependent. Taken together, these data suggest that the 76 most NH(2)-terminal residues (ULD) dramatically regulate the protein levels of parkin.
Finney
Natalie
N
Nervous System Research, Novartis Pharma AG, CH-4002 Basel, Switzerland.
Walther
Fabienne
F
Mantel
Pierre-Yves
PY
Stauffer
Daniela
D
Rovelli
Giorgio
G
Dev
Kumlesh K
KK
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D006801:Humans; D008025:Ligases / Q000032:analysis* / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000502:physiology*; D013329:Structure-Activity Relationship; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D010446:Peptide Fragments; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D008025:Ligases