Limits...
The principal PINK1 and Parkin cellular events triggered in response to dissipation of mitochondrial membrane potential occur in primary neurons.

Koyano F, Okatsu K, Ishigaki S, Fujioka Y, Kimura M, Sobue G, Tanaka K, Matsuda N - Genes Cells (2013)

Bottom Line: We found that dissipation of the mitochondrial membrane potential triggers phosphorylation of both PINK1 and Parkin and that, in response, Parkin translocates to depolarized mitochondria.Furthermore, Parkin's E3 activity is re-established concomitant with ubiquitin-ester formation at Cys431 of Parkin.As a result, mitochondrial substrates in neurons become ubiquitylated.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.

Show MeSH

Related in: MedlinePlus

Several outer membrane mitochondrial proteins underwent Parkin-dependent ubiquitylation after a decrease in the membrane potential. (A) Ubiquitin–oxyester formation on Parkin (shown by the red asterisk) was specifically observed in the Parkin C431S mutant after CCCP treatment in primary neurons. This modification was not observed in wild-type Parkin or the C431F mutant. (B) Intact primary neurons, or primary neurons infected with lentivirus encoding Parkin, were treated with CCCP and then immunoblotted to detect endogenous Mfn2, Miro1, HKI, VDAC1, Mfn1, Tom70 and Tom20. The red arrowheads and asterisks indicate ubiquitylated proteins. (C) Ubiquitylation of Mfn2 after mitochondrial depolarization (shown by the red asterisk) is prevented by PARKIN knockout in primary neurons.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3842116&req=5

fig04: Several outer membrane mitochondrial proteins underwent Parkin-dependent ubiquitylation after a decrease in the membrane potential. (A) Ubiquitin–oxyester formation on Parkin (shown by the red asterisk) was specifically observed in the Parkin C431S mutant after CCCP treatment in primary neurons. This modification was not observed in wild-type Parkin or the C431F mutant. (B) Intact primary neurons, or primary neurons infected with lentivirus encoding Parkin, were treated with CCCP and then immunoblotted to detect endogenous Mfn2, Miro1, HKI, VDAC1, Mfn1, Tom70 and Tom20. The red arrowheads and asterisks indicate ubiquitylated proteins. (C) Ubiquitylation of Mfn2 after mitochondrial depolarization (shown by the red asterisk) is prevented by PARKIN knockout in primary neurons.

Mentions: Klevit's group recently reported that Cys357 in the RING2 domain of RBR-type E3 HHARI is an active catalytic residue and forms an ubiquitin–thioester intermediate during ubiquitin ligation (Wenzel et al. 44). Parkin is also a RBR-type E3 with Parkin Cys431 equivalent to HHARI Cys357. We and a number of groups recently independently showed that a Parkin C431S mutant forms a stable ubiquitin–oxyester on CCCP treatment in non-neuronal cell lines, suggesting the formation of an ubiquitin–thioester intermediate (Lazarou et al. 20) (M.I., K.T., and N.M., unpublished data). To examine whether Parkin forms an ubiquitin–ester intermediate in neurons as well, we again used a lentivirus to express HA-Parkin with the C431S mutation, which converts an unstable ubiquitin–thioester bond to a stable ubiquitin–oxyester bond. The HA-Parkin C431S mutant specifically exhibited an upper-shifted band equivalent to an ubiquitin–adduct after CCCP treatment (Fig. 4A, lane 4). This modification was not observed in wild-type HA-Parkin (lane 2) and was absent when an ester-deficient pathogenic mutation, C431F, was used (lane 6), suggesting ubiquitin–oxyester formation of Parkin when neurons are treated with CCCP.


The principal PINK1 and Parkin cellular events triggered in response to dissipation of mitochondrial membrane potential occur in primary neurons.

Koyano F, Okatsu K, Ishigaki S, Fujioka Y, Kimura M, Sobue G, Tanaka K, Matsuda N - Genes Cells (2013)

Several outer membrane mitochondrial proteins underwent Parkin-dependent ubiquitylation after a decrease in the membrane potential. (A) Ubiquitin–oxyester formation on Parkin (shown by the red asterisk) was specifically observed in the Parkin C431S mutant after CCCP treatment in primary neurons. This modification was not observed in wild-type Parkin or the C431F mutant. (B) Intact primary neurons, or primary neurons infected with lentivirus encoding Parkin, were treated with CCCP and then immunoblotted to detect endogenous Mfn2, Miro1, HKI, VDAC1, Mfn1, Tom70 and Tom20. The red arrowheads and asterisks indicate ubiquitylated proteins. (C) Ubiquitylation of Mfn2 after mitochondrial depolarization (shown by the red asterisk) is prevented by PARKIN knockout in primary neurons.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842116&req=5

fig04: Several outer membrane mitochondrial proteins underwent Parkin-dependent ubiquitylation after a decrease in the membrane potential. (A) Ubiquitin–oxyester formation on Parkin (shown by the red asterisk) was specifically observed in the Parkin C431S mutant after CCCP treatment in primary neurons. This modification was not observed in wild-type Parkin or the C431F mutant. (B) Intact primary neurons, or primary neurons infected with lentivirus encoding Parkin, were treated with CCCP and then immunoblotted to detect endogenous Mfn2, Miro1, HKI, VDAC1, Mfn1, Tom70 and Tom20. The red arrowheads and asterisks indicate ubiquitylated proteins. (C) Ubiquitylation of Mfn2 after mitochondrial depolarization (shown by the red asterisk) is prevented by PARKIN knockout in primary neurons.
Mentions: Klevit's group recently reported that Cys357 in the RING2 domain of RBR-type E3 HHARI is an active catalytic residue and forms an ubiquitin–thioester intermediate during ubiquitin ligation (Wenzel et al. 44). Parkin is also a RBR-type E3 with Parkin Cys431 equivalent to HHARI Cys357. We and a number of groups recently independently showed that a Parkin C431S mutant forms a stable ubiquitin–oxyester on CCCP treatment in non-neuronal cell lines, suggesting the formation of an ubiquitin–thioester intermediate (Lazarou et al. 20) (M.I., K.T., and N.M., unpublished data). To examine whether Parkin forms an ubiquitin–ester intermediate in neurons as well, we again used a lentivirus to express HA-Parkin with the C431S mutation, which converts an unstable ubiquitin–thioester bond to a stable ubiquitin–oxyester bond. The HA-Parkin C431S mutant specifically exhibited an upper-shifted band equivalent to an ubiquitin–adduct after CCCP treatment (Fig. 4A, lane 4). This modification was not observed in wild-type HA-Parkin (lane 2) and was absent when an ester-deficient pathogenic mutation, C431F, was used (lane 6), suggesting ubiquitin–oxyester formation of Parkin when neurons are treated with CCCP.

Bottom Line: We found that dissipation of the mitochondrial membrane potential triggers phosphorylation of both PINK1 and Parkin and that, in response, Parkin translocates to depolarized mitochondria.Furthermore, Parkin's E3 activity is re-established concomitant with ubiquitin-ester formation at Cys431 of Parkin.As a result, mitochondrial substrates in neurons become ubiquitylated.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.

Show MeSH
Related in: MedlinePlus