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Mechanism of human PINK1 activation at the TOM complex in a reconstituted system

SCIENCE ADVANCES. 2024-06; 
Olawale G Raimi, Hina Ojha, Kenneth Ehses, Verena Dederer, Sven M Lange, Cristian Polo Rivera, Tom D Deegan, Yinchen Chen, Melanie Wightman, Rachel Toth, Karim P M Labib, Sebastian Mathea, Neil Ranson, Rubén Fernández-Busnadiego, Miratul M K Muqit
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Proteins, Expression, Isolation and Analysis For cloning of hPINK1 constructs for insect cell expression, the coding sequences for the PINK1 constructs were polymerase chain reaction (PCR)–amplified using clone OHu25380D (GenScript) as a template and cloned into the vector pFB-6HZB [Structural Genomics Consortium (SGC); Addgene ID: 218680] as previously described. Get A Quote

摘要

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of early-onset Parkinson's disease (PD). Stabilization of PINK1 at the translocase of outer membrane (TOM) complex of damaged mitochondria is critical for its activation. The mechanism of how PINK1 is activated in the TOM complex is unclear. Here, we report that co-expression of human PINK1 and all seven TOM subunits in Saccharomyces cerevisiae is sufficient for PINK1 activation. We use this reconstitution system to systematically assess the role of each TOM subunit toward PINK1 activation. We unambiguously demonstrate that the TOM20 and TOM70 receptor subunits are required for optimal PINK1 activation and map their sites of intera... More

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