Structural Characterization of Endogenous Tuberous Sclerosis Protein Complex Revealed Potential Polymeric Assembly

Tuberous sclerosis protein complex
(pTSC) nucleates a proteinaceous
signaling hub that integrates information about the internal and external
energy status of the cell in the regulation of growth and energy consumption.
Biochemical and cryo-electron microscopy studies of recombinant pTSC
have revealed its structure and stoichiometry and hinted at the possibility
that the complex may form large oligomers. Here, we have partially
purified endogenous pTSC from fasted mammalian brains of rat and pig
by leveraging a recombinant antigen binding fragment (Fab) specific for the TSC2 subunit of pTSC. We demonstrate Fab-dependent purification of pTSC from membrane-solubilized fractions
of the brain homogenates. Negative stain electron microscopy of the
samples purified from pig brain demonstrates rod-shaped protein particles
with a width of 10 nm, a variable length as small as 40 nm, and a
high degree of conformational flexibility. Larger filaments are evident
with a similar 10 nm width and a ≤1 μm length in linear
and weblike organizations prepared from pig brain. Immunogold labeling
experiments demonstrate linear aggregates of pTSC purified from mammalian
brains. These observations suggest polymerization of endogenous pTSC
into filamentous superstructures.