Stoichiometry and assembly of mTOR complexes revealed by single-molecule pulldown

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• The mammalian target of rapamycin (mTOR) kinase is a crucial regulator of cellular, developmental, and metabolic processes.
• Dysregulation of mTOR signaling is linked to diseases like cancer and diabetes.
• The molecular mechanisms of mTOR complex (mTORC) assembly and oligomerization remain largely unknown.

Purpose of the Study:

• To investigate the stoichiometry and assembly of mTORC1 and mTORC2 complexes.
• To elucidate the oligomeric state of mTORCs using a novel single-molecule approach.
• To understand how physiological conditions and drug treatments affect mTORC assembly.

Main Methods:

• Utilized the single-molecule pulldown (SiMPull) assay, combining pulldown techniques with single-molecule fluorescence microscopy.
• Validated the SiMPull assay with mTORC1, confirming its previously reported dimeric assembly.
• Applied SiMPull to analyze the stoichiometry and assembly of mTORC2 components.

Main Results:

• mTORC2 major components exist in two copies per complex, indicating homodimeric assembly.
• Individual mTORC subunits are monomeric when free, with dimerization arising from multiple subunits forming a composite surface.
• Nutrient or energy stress did not alter mTORC stoichiometry, while rapamycin transiently produced monomeric mTORC1 before complex disruption, leaving mTORC2 unaffected.

Conclusions:

• mTORC1 and mTORC2 likely assemble as homodimers through interactions involving multiple subunits.
• SiMPull assay effectively distinguishes complex disassembly from stoichiometry changes.
• These findings provide critical insights into mTORC assembly, potentially guiding future therapeutic strategies.