Cytoplasmic localization and proteasomal degradation of N-terminally cleaved form of PINK1

doi:10.1016/j.neulet.2007.10.019

Neuroscience Letters

Volume 430, Issue 1, 3 January 2008, Pages 13-17

https://doi.org/10.1016/j.neulet.2007.10.019 Get rights and content

Abstract

Mutations in PTEN-induced putative kinase 1 (PINK1) gene have been linked to an autosomal recessive form of familial Parkinson's disease. PINK1 encodes a predicted mitochondrial protein kinase. Although the mitochondrial localization of PINK1 has been suggested, the exact subcellular compartment in which PINK1 exerts its cytoprotective function is elusive. Thus, we studied the subcellular distribution and metabolism of PINK1 in cultured cells. Immunocytochemical analysis showed that PINK1 resides in cytoplasm in addition to mitochondria, and that the mitochondrial localization is dependent on its N-terminal sequence. Cellular expression of PINK1 yielded several N-terminally cleaved fragments as well as the full-length protein, among which the 54 kDa fragment (ΔN 54 kDa) was highly accumulated in the presence of proteasome inhibitors. Endogenous PINK1 was detected dominantly in the form of ΔN 54 kDa upon proteasome inhibition. Rapid turnover of ΔN 54 kDa further supported its higher susceptibility to proteasomal degradation compared with that of full-length protein. These results indicate that ΔN 54 kDa PINK1 undergoes constitutive degradation by proteasome, and underscore the significance of its localization in cytoplasm, especially in the N-terminally processed form.

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Acknowledgements

The authors thank Drs. M. Unoki and Y. Nakamura for PINK1 cDNA, and Dr. M. Miyagishi for technical advices in siRNA design.

References (18)

W. Dauer et al.
Parkinson's disease: mechanisms and models
Neuron
(2003)
M. Ditzel et al.
IAP degradation: decisive blow or altruistic sacrifice?
Trends Cell Biol.
(2002)
A. Petit et al.
Wild-type PINK1 prevents basal and induced neuronal apoptosis, a protective effect abrogated by Parkinson's disease-related mutations
J. Biol. Chem.
(2005)
P.M. Abou-Sleiman et al.
Expanding insights of mitochondrial dysfunction in Parkinson's disease
Nat. Rev. Neurosci.
(2006)
A. Beilina et al.
Mutations in PTEN-induced putative kinase 1 associated with recessive parkinsonism have differential effects on protein stability
Proc. Natl. Acad. Sci. U.S.A.
(2005)
I.E. Clark et al.
Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin
Nature
(2006)
S.M. Elbashir et al.
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells
Nature
(2001)
S. Gandhi et al.
PINK1 protein in normal human brain and Parkinson's disease
Brain
(2006)
T. Kitada et al.
From the cover: impaired dopamine release and synaptic plasticity in the striatum of PINK1-deficient mice
Proc. Natl. Acad. Sci. U.S.A.
(2007)

There are more references available in the full text version of this article.

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Cytoplasmic localization and proteasomal degradation of N-terminally cleaved form of PINK1

Abstract

Section snippets

Acknowledgements

Neuron

Trends Cell Biol.

J. Biol. Chem.

Expanding insights of mitochondrial dysfunction in Parkinson's disease

Nat. Rev. Neurosci.

Mutations in PTEN-induced putative kinase 1 associated with recessive parkinsonism have differential effects on protein stability

Proc. Natl. Acad. Sci. U.S.A.

Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin

Nature

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Nature

PINK1 protein in normal human brain and Parkinson's disease

Brain

From the cover: impaired dopamine release and synaptic plasticity in the striatum of PINK1-deficient mice

Proc. Natl. Acad. Sci. U.S.A.