"Inside-in" or "inside-out"? The membrane topology of SLC41A1.

"Inside-in" or "inside-out"? The membrane topology of SLC41A1. Magnes Res. 2014 Feb 3; Authors: Sponder G, Rutschmann K, Kolisek M Abstract Membrane topology is an important parameter for understanding the function and regulation of any integral protein. This aspect of the NME SLC41A1 is currently under debate. The most probable model, which has been computer-predicted, exhibits ten TMh with both termini being oriented intracellularly. However, other freely accessible online prediction programs predict that SLC41A1 possesses eleven ("outside-in" configuration), nine ("outside-in" configuration), or eight ("inside-in" configuration) TMh. The consensus based on published experimental data acquired by independent research teams is that the N-terminal flanking region is located intracellularly. However, controversy remains about the orientation of the C-terminus, which has lately been proposed to be extracellular in peer-reviewed bibliography. Here, we performed split-ubiquitin functional assays with transgenic SLC41A1 fused N- or C-terminally to a Cub-LexA-VP16 reporter cassette. The bait constructs were co-expressed in S. cerevisiae st. NMY51 with positive recombinant membrane markers (Ost1, Fur4, Alg5, Tom20) tagged with NubI (or NubG). Ubiquitin could only be reconstituted if the reporter moiety was exposed to the cytosol. Functional reconstitution of ubiquitin was observed when SLC41A1 C-terminally tagged with Cub was co-expressed with NubI-...
Source: Magnesium Research - Category: Biochemistry Authors: Tags: Magnes Res Source Type: research