Disrupting Mitochondrial Homeostasis via TBC1D15 Protein
TL;DR
This work resolves the long-standing ambiguity between TBC1D15 total expression level and its subcellular shuttling, establishing a new conceptual framework that prioritizes protein translocation over bulk abundance in organelle quality control.
Abstract
Highlights What are the main findings? Subcellular inter-organelle trafficking of TBC1D15, rather than its whole-cell protein abundance, is identified as the critical switch governing mitochondria–lysosome homeostasis and mitochondrial quality control. CCCP and high-dose AuNPs modulate TBC1D15 through two divergent regulatory modes: CCCP induces a PINK1-independent biphasic TBC1D15 fluctuation, whereas AuNPs trap TBC1D15 on the mitochondrial outer membrane and block its lysosomal delivery via a PINK1-independent route. What are the implications of the main findings? This work resolves the long-standing ambiguity between TBC1D15 total expression level and its subcellular shuttling, establishing a new conceptual framework that prioritizes protein translocation over bulk abundance in organelle quality control. High-dose AuNPs can be exploited as a specific pharmacological inhibitor of TBC1D15 trafficking, offering a potential bypass strategy and therapeutic clues for neurodegenerative and metabolic disorders characterized by defective PINK1/Parkin-mediated mitophagy. Abstract Background: Mitochondria–lysosome coupling and mitophagy are fundamental to mitochondrial homeostasis, yet the determinants governing this process remain incompletely defined. This study distinguished the regulatory modes of TBC1D15 and established its functional role as a downstream effector in mitochondria-linked autophagy. Methods: PC-12 and HeLa cells were treated with CCCP, gradient-concentrated AuNPs, and CQ, combined with siRNA-mediated PINK1 knockdown. Whole-cell, mitochondrial and lysosomal fractions were analyzed by immunoblotting. Results: Inter-organelle translocation of TBC1D15, rather than its total abundance, serves as the critical switch for mitochondria–lysosome homeostasis. CCCP induced a time-dependent biphasic change in TBC1D15 that persisted upon PINK1 knockdown, indicating regulation is not strictly PINK1-dependent. High-dose AuNPs trapped TBC1D15 on mitochondria and blocked lysosomal delivery via a PINK1-independent route. AuNPs exhibited biphasic concentration-dependent effects: low concentrations moderately upregulated TBC1D15, promoting adaptive organelle contacts, while millimolar concentrations caused pathological tethering. Subcellular fractionation and CQ intervention confirmed that defective lysosomal delivery underlies mitochondrial TBC1D15 accumulation upon AuNP challenge. Conclusions: This work establishes two distinct regulatory axes converging on TBC1D15. High-dose AuNPs can serve as a specific pharmacological blocker of TBC1D15 trafficking, offering therapeutic avenues for disorders characterized by impaired mitophagy.