TBC1D4 antagonizes RAB2A-mediated autophagic and endocytic pathways

Area of Science:

• Cell Biology
• Molecular Biology
• Physiology

Background:

• Autophagic and endocytic pathways are crucial for cellular homeostasis.
• The coordination of these pathways for lysosomal degradation is not fully understood.
• RAB2A GTPase regulates both autophagic and endocytic processes.

Purpose of the Study:

• To investigate the role of TBC1D4 (TBC1 domain family member 4) in regulating RAB2A-mediated autophagy and endocytosis.
• To elucidate the molecular mechanisms by which TBC1D4 controls these pathways.
• To assess the in vivo consequences of TBC1D4 deficiency in relevant tissues.

Main Methods:

• Co-immunoprecipitation assays to study protein interactions.
• Western blotting to analyze protein levels and complex formation.
• Autophagy and endocytosis assays in cell culture and knockout mouse models.
• Confocal microscopy and correlative light electron microscopy (CLEM).

Main Results:

• TBC1D4 binds to RAB2A, inhibiting early-stage autophagy by preventing ULK1 complex activation.
• TBC1D4 interacts with RUBCNL, disrupting the autophagosomal complex (RAB2A, RUBCNL, STX17) and impairing autophagosome-lysosome fusion.
• TBC1D4 inhibits RAB2A-mediated endocytic degradation independently of RUBCNL.
• Hepatocyte- or adipocyte-specific TBC1D4 knockout mice exhibit increased autophagic flux, enhanced endocytic degradation, and tissue damage.

Conclusions:

• TBC1D4 acts as a critical molecular brake, suppressing both autophagic and endocytic pathways.
• TBC1D4 and RAB2A form a dual molecular switch regulating lysosomal degradation.
• Dysregulation of TBC1D4 leads to detrimental effects on tissue homeostasis in vivo.