Transport assays using purified glucose transporters (GLUTs) have proven to be difficult to implement, hampering deeper mechanistic insights. Here the authors have optimized a transport assay in liposomes that will provide insight to study other membrane transport proteins. Glucose transporters (GLUTs) are essential for organism-wide glucose homeostasis in mammals, and their dysfunction is associated with numerous diseases, such as diabetes and cancer. Despite structural advances, transport assays using purified GLUTs have proven to be difficult to implement, hampering deeper mechanistic insights. Here, we have optimized a transport assay in liposomes for the fructose-specific isoform GLUT5. By combining lipidomic analysis with native MS and thermal-shift assays, we replicate the GLUT5 transport activities seen in crude lipids using a small number of synthetic lipids. We conclude that GLUT5 is only active under a specific range of membrane fluidity, and that human GLUT1-4 prefers a similar lipid composition to GLUT5. Although GLUT3 is designated as the high-affinity glucose transporter, in vitro D-glucose kinetics demonstrates that GLUT1 and GLUT3 actually have a similar K-M,K- but GLUT3 has a higher turnover. Interestingly, GLUT4 has a high K-M for D-glucose and yet a very slow turnover, which may have evolved to ensure uptake regulation by insulin-dependent trafficking. Overall, we outline a much-needed transport assay for measuring GLUT kinetics and our analysis implies that high-levels of free fatty acid in membranes, as found in those suffering from metabolic disorders, could directly impair glucose uptake.

The Na⁺/H⁺ exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na⁺/Li⁺ exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite the functional importance of NHA2, structural information and the molecular basis for its ion-exchange mechanism have been lacking. Here we report the cryo-EM structures of bison NHA2 in detergent and in nanodiscs, at 3.0 and 3.5 Å resolution, respectively. The bison NHA2 structure, together with solid-state membrane-based electrophysiology, establishes the molecular basis for electroneutral ion exchange. NHA2 consists of 14 transmembrane (TM) segments, rather than the 13 TMs previously observed in mammalian Na⁺/H⁺ exchangers (NHEs) and related bacterial antiporters. The additional N-terminal helix in NHA2 forms a unique homodimer interface with a large intracellular gap between the protomers, which closes in the presence of phosphoinositol lipids. We propose that the additional N-terminal helix has evolved as a lipid-mediated remodeling switch for the regulation of NHA2 activity. 

Solute carrier transporters (SLCs) mediate the inter- and intra- cellular trafficking of a plethora of substrates and are essential to cell homeostasis. Despite their importance to human physiology and their potential as therapeutic targets, many SLCs are considered orphans as the physiological substrate has not been experimentally determined. Furthermore, SLCs remain understudied and underutilized, partly due to the inherent difficulties in working with SLC transporters. In this thesis, I present the development of the GFP-based thermostability assay (GFP-TS), which enables the detection of ligand-SLC interactions using un-purified material, but with the same high-throughput screening capability as dye-based thermal-shift assays. We highlight how GFP-TS is an excellent complement to native mass spectrometry approaches for analyzing lipid-protein interactions, by demonstrating how specific lipids modulate oligomerization in Na+/H+ exchangers. GFP-TS was combined with other biochemical approaches to show that not all SLC35 members transport nucleotide-sugars, as currently believed. Specifically, we unequivocally demonstrate that SLC35B1 is a strict ADP/ATP exchanger, which is critical for cell homeostasis, as it supplies the endoplasmic reticulum (ER) with ATP. Finally, I outline the progress towards elucidating the function of the synaptic vesicle protein 2A (SV2A), an enigmatic brain SLC transporter that is the receptor for clinically-used anti-epileptic drugs. In summary, my research contributes to the growing body of knowledge of SLC function, and outlines how a simple thermal stability can be utilised for determining ligand- and lipid-interactions of SLC transporters.

Membrane bilayers are made up of a myriad of different lipids that regulate the functional activity, stability, and oligomerization of many membrane proteins. Despite their importance, screening the structural and functional impact of lipid-protein interactions to identify specific lipid requirements remains a major challenge. Here, we use the FSEC-TS assay to show cardiolipin-dependent stabilization of the dimeric sodium/proton antiporter NhaA, demonstrating its ability to detect specific protein-lipid interactions. Based on the principle of FSECTS, we then engineer a simple thermal-shift assay (GFP-TS), which facilitates the highthroughput screening of lipid-and ligand-interactions with membrane proteins. By comparing the thermostability of medically relevant eukaryotic membrane proteins and a selection of bacterial counterparts, we reveal that eukaryotic proteins appear to have evolved to be more dependent to the presence of specific lipids.