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

Christoph Bock,Katrin Reichel,Franz Tumulka,Rupert Abele,Volker Dötsch,Robert Tampé,Gerhard Hummer,Benesh Joseph,Lars Schäfer,Frank Löhr,Julia Würz,Frank Bernhard

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.

Highlights

ATP-binding cassette (ABC) proteins represent the largest family of primary active membrane transport proteins[1]
Human embryonic kidney (HEK) 293 T cells were transiently transfected with expression constructs of TMD0-myc or cf-TMD0-myc alone or together with coreTAPL and lysed after 48 h with the mild detergent digitonin
The TMD0 construct designed for cf expression behaves as wt TMD0 in respect of expression yield in HEK 293 T cells, interaction with coreTAPL, and oligomerization

Summary

Introduction

ATP-binding cassette (ABC) proteins represent the largest family of primary active membrane transport proteins[1]. Members of the ABCC family represent full-size transporters, some of which harbor an extra five TMHs containing N-terminal TMD04. In the heterodimeric complex TAP, composed of TAP1 and TAP2, the TMD0s interact with tapasin via a conserved salt bridge in the center of the endoplasmic reticulum membrane[6]. The homodimeric half-transporter TAPL comprises two functional units (Fig. 1): (1) coreTAPL (residue 143 to 766), consisting of two conserved NBDs and 2 × 6 TMHs, is fully functional in peptide transport; (2) the N-terminal transmembrane domain TMD0 (residue 1 to 142). TMD0 displays an interaction platform with the lysosomal associated membrane proteins LAMP-1 and LAMP-2B. This interaction extends the half-life of TAPL by a factor of five[14]. The topology of TMD0 was confirmed by paramagnetic relaxation enhancement (PRE) and by nuclear Overhauser effects (NOEs)

Methods

Results

Conclusion