Assembly and molecular architecture of the TSC1-TSC2 complex

Dipanjan Guha¹, Jurgen Plitzko², Mark Nellist³, Daniel Nemecek^1,2

¹Central European Institute of Technology, Masaryk University, Brno, CZ

²Department of Biochemistry, Max Planck Institute, Martinsried, DE

³Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, NL

The TSC1-TSC2 complex plays an important role in the mechanistic target of rapamycin (mTOR) signalling pathway that integrates signals from extracellular growth factors, nutrients, energy deficit or inflammation and downstream controls cell metabolism and growth [1]. TSC1 is a ~130 kDa hydrophilic protein that shows no sequence homology to other known vertebrate proteins and likely guides and stabilizes the proper assembly of the TSC1-TSC2 complex [3]. TSC2 is a ~200 kDa protein that contains a conserved 163 amino acid region close to the C-terminus that is homologous to GTPase activating proteins (GAPs). The TSC1-TSC2 complex possesses RHEB GAP activity [4], and conversion of active GTP-bound RHEB into the inactive GDP-bound form by the complex downregulates the activity of mTOR complex 1 (TORC1) [1]. Pathogenic mutations in either TSC1) or TSC2 lead to tuberous sclerosis complex (TSC), an autosomal dominant disorder characterized by neurological symptoms, skin and renal abnormalities [2]. Recently, it has been shown that TSC1 and TSC2 assemble into high molecular weight complexes (>1 MDa) [5,6]. However, the molecular architecture of this complex is unknown. We co-expressed epitope-tagged TSC1 and TSC2 in HEK 293T cells and imaged the affinity purified TSC1-TSC2 complexes by negative stain electron microscopy. Micrographs showed small rings of the complex that had a disk-like appearance. Initial alignments and classification of ~1000 particles revealed two kinds of rings: a smaller ring with ~70-Å diameter that seems to be composed of 5 subunits and a larger ~90-Å ring structure that may correspond to a heteroctamer of four TSC1 and four TSC2 subunits, according to the estimated average molecular mass of the complex. Ongoing image analysis aims to identify the different subunits in the two respective complexes and provide the structural basis for their function.

[1] Laplante M, Sabatini DM (2012) Cell, 149, 274.

[2] Gomez, M., et al. (2013) The tuberous sclerosis complex, Oxford University Press.

[3] Sun, W., et al. (2013) Nat.Commun., 4, 2135.

[4] Maheshwar, M. M., et al. (1997) Hum.Mol.Genet., 6, 1991.

[5] Hoogeveen-Westerveld, M., et al. (2012) BMC Biochem., 13, 18.

[6] Menon S., et al. (2014) Cell 156, 771.