mTOR-mediated phosphorylation of VAMP8 and SCFD1 regulates autophagosome maturation

Area of Science:

• Cell Biology
• Molecular Biology
• Autophagy Research

Background:

• The mammalian target of rapamycin (mTORC1) pathway is a key regulator of cellular processes, including autophagy.
• Autophagy is crucial for cellular homeostasis, involving the degradation of damaged components via autolysosomes.
• The precise mechanisms by which mTORC1 influences autophagosome-lysosome fusion, particularly concerning SNARE complex regulation, are not fully understood.

Purpose of the Study:

• To elucidate the role of mTORC1 in regulating the N-ethylmaleimide-sensitive protein receptor (SNARE) complex during autophagy.
• To investigate how mTORC1-mediated signaling affects the formation of the STX17-SNAP29-VAMP8 SNARE complex.
• To determine the impact of VAMP8 phosphorylation on autophagosome-lysosome fusion and related cellular processes.

Main Methods:

• Phosphorylation analysis of VAMP8 by mTORC1.
• In vitro assays using VAMP8 phosphorylation mimic mutants to assess autophagosome-lysosome fusion.
• Identification and characterization of SCFD1's role in SNARE complex formation and autophagosome-lysosome fusion.
• Assessment of VAMP8 dephosphorylation-dependent SCFD1 recruitment to autolysosomes.
• In vivo studies using mouse liver models to evaluate the physiological consequences of VAMP8 phosphorylation.

Main Results:

• mTORC1 directly inhibits the formation of the STX17-SNAP29-VAMP8 SNARE complex by phosphorylating VAMP8.
• Phosphorylation of VAMP8 by mTORC1 prevents autophagosome-lysosome fusion.
• A phosphorylation mimic mutant of VAMP8 failed to promote autophagosome-lysosome fusion in vitro.
• SCFD1, a Sec1/Munc18-like protein, is identified as essential for SNARE complex assembly and autophagosome-lysosome fusion at the autolysosome.
• Dephosphorylated VAMP8 facilitates SCFD1 recruitment to autolysosomes, promoting fusion; conversely, phosphorylated VAMP8 or SCFD1 depletion inhibits fusion.
• Expression of phosphomimic VAMP8 in mouse liver led to increased lipid droplet accumulation, suggesting impaired autophagy.

Conclusions:

• mTORC1-mediated phosphorylation of VAMP8 serves as a critical inhibitory mechanism for autophagosome-lysosome fusion.
• The phosphorylation status of VAMP8 dictates its interaction with SCFD1, thereby controlling SNARE complex formation and fusion progression.
• This regulatory axis highlights a novel link between mTORC1 signaling and the machinery governing autophagosome-lysosome fusion, with implications for cellular lipid metabolism.