Parkin is associated with cellular vesicles.
We recently identified a novel gene, parkin, as a pathogenic gene for autosomal recessive juvenile parkinsonism. Parkin encodes a 52-kDa protein with a ubiquitin-like domain and two RING-finger motifs. To provide a insight into the function of parkin, we have examined its intracellular distribution in cultured cells. We found that parkin was localized in the trans-Golgi network and the secretory vesicles in U-373MG or SH-SY5Y cells by immunocytochemical analyses. In the subsequent subcellular fractionation studies of rat brain, we showed that parkin was copurified with the synaptic vesicles (SVs) when we used low ionic conditions throughout the procedure. An immunoelectromicroscopic analysis indicated that parkin was present on the SV membrane. Parkin was readily released from SVs into the soluble phase by increasing ionic strength at neutral pH, but not by a non-ionic detergent. To elucidate its responsible region for membrane association, we transfected with green fluorescent protein-tagged deletion mutants of parkin into COS-1 cells followed by subcellular fractionation. We demonstrated the ability of parkin to bind to the membranes through a broad region except for the ubiquitin-like domain. The significance of SV localization of parkin is discussed.
Kubo
S I
SI
Department of Neurology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo, Tokyo 113-8421, Japan.
Kitami
T
T
Noda
S
S
Shimura
H
H
Uchiyama
Y
Y
Asakawa
S
S
Minoshima
S
S
Shimizu
N
N
Mizuno
Y
Y
Hattori
N
N
D000818:Animals; D019677:Artificial Gene Fusion; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000502:physiology; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D008025:Ligases