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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.
Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na+ across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430 kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS.