2. Academic Validation
  3. Structural and functional insights into the interaction and targeting hub TMD0 of the polypeptide transporter TAPL

Structural and functional insights into the interaction and targeting hub TMD0 of the polypeptide transporter TAPL

  • Sci Rep. 2018 Oct 23;8(1):15662. doi: 10.1038/s41598-018-33841-w.
Christoph Bock 1,  Frank Löhr 2,  Franz Tumulka 1,  Katrin Reichel 3,  Julia Würz 2,  Gerhard Hummer 4,  Lars Schäfer 5,  Robert Tampé 1,  Benesh Joseph 3,  Frank Bernhard 2,  Volker Dötsch 2,  Rupert Abele 6
Affiliations

Affiliations

  • 1 Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Max-von-Laue Str. 9, 60438, Frankfurt am Main, Germany.
  • 2 Institute of Biophysical Chemistry & Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Max-von-Laue Str. 9, 60438, Frankfurt am Main, Germany.
  • 3 Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue Str. 9, 60438, Frankfurt am Main, Germany.
  • 4 Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Max-von-Laue Str. 3, 60438, Frankfurt am Main, Germany.
  • 5 Lehrstuhl für Theoretische Chemie, Ruhr-University Bochum, 4780, Bochum, Germany.
  • 6 Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Max-von-Laue Str. 9, 60438, Frankfurt am Main, Germany. [email protected].
PMID: 30353140 DOI: 10.1038/s41598-018-33841-w
Abstract

The ATP-binding cassette transporter TAPL translocates polypeptides from the cytosol into the lysosomal lumen. TAPL can be divided into two functional units: coreTAPL, active in ATP-dependent peptide translocation, and the N-terminal membrane spanning domain, TMD0, responsible for cellular localization and interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. Although the structure and function of ABC transporters were intensively analyzed in the past, the knowledge about accessory membrane embedded domains is limited. Therefore, we expressed the TMD0 of TAPL via a cell-free expression system and confirmed its correct folding by NMR and interaction studies. In cell as well as cell-free expressed TMD0 forms oligomers, which were assigned as dimers by PELDOR spectroscopy and static LIGHT scattering. By NMR spectroscopy of uniformly and selectively isotope labeled TMD0 we performed a complete backbone and partial side chain assignment. Accordingly, TMD0 has a four transmembrane helix topology with a short helical segment in a lysosomal loop. The topology of TMD0 was confirmed by paramagnetic relaxation enhancement with paramagnetic stearic acid as well as by nuclear Overhauser effects with c6-DHPC and cross-peaks with water.

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