Sub-Nanometer Cryo-EM Density Map of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2

Abstract

Heterodimeric amino acid transporters (HATs) are protein complexes mediating the transport of amino acids and derivatives thereof across biological membranes. HATs are composed of two subunits, a heavy and a light chain subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. The human HAT 4F2hc-LAT2 is composed of the type-II membrane N-glycoprotein 4F2hc (SCL3A2) and the L-type amino acid transporter LAT2 (SLC7A8), which are covalently linked to each other by a conserved disulfide bridge. Whereas LAT2 catalyzes substrate transport, 4F2hc is important for the successful trafficking of the transporter to the plasma membrane. The overexpression, malfunction, or absence of 4F2hc-LAT2 is associated with human diseases, and therefore, this heterodimeric complex represents a potential drug target. The recombinant human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast Pichia pastoris, and the protein can be purified. Here, we present the cryo-EM density map of the human 4F2hc-LAT2 amino acid transporter at sub-nanometer resolution. A homology model of 4F2hc-LAT2 in the inward-open conformation was generated and fitted into the cryo-EM density and analyzed. In addition, disease-causing point mutations in human LAT2 were mapped on the homology model of 4F2hc-LAT2, and the possible functional implications on the molecular level are discussed.

Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein

“…The chromatogram of 4F2hc-LAT1 displayed a prominent, sharp peak, indicating that the double affinity purification procedure applied yielded a pure and monodisperse membrane protein sample (Figure 3A). The elution volume was similar to the GDN-solubilized heterodimeric complex of 4F2hc-LAT2, as previously reported [16]. Since 4F2hc-LAT1 and 4F2hc-LAT2 share a high sequence similarity of 48% and are structurally similar [14,16,20], these data, together with the results obtained from SDS-PAGE and Western blot analysis (Figure 2) further, corroborated correct assembly of the heterodimeric complex 4F2hc-LAT1.…”

“…In addition, negligible contributions of mainly 4F2hc monomers were discerned (Figure 2, lane 1). The latter observation of minor amounts of free 4F2hc and also possibly LAT1 might reflect SDS-based denaturation of the complex due to SDS-PAGE, which was previously observed by SDS-and blue native PAGE, and corroborated by size exclusion chromatography (SEC) of 4F2hc-LAT2 [16,19]. Western blots probed with either αHis (Figure 2, lane 3) or αStrep antibodies (Figure 2, lane 5) under non-reducing conditions confirmed the high purity and homogeneity of 4F2hc-LAT1 by also displaying prominent bands at 140 kDa in either case.…”

“…We made use of the His-tag of 4F2hc first and bound the protein to a Ni-NTA resin. Due to the stabilizing effect of steroid-based detergents or mixtures thereof on system L HATs [18,22], and for the sake of convenience to compare results obtained from this study to previously reported ones [14,16], we exchanged the detergent mixture LMNG/CHS with glyco-diosgenin (GDN) during this step of protein purification. After protein elution from the Ni-NTA resin, partially pure 4F2hc-LAT1 could be isolated.…”

“…4F2hc can associate with selected light subunits to form HATs belonging to different transport systems [3]. System L is comprised of two HATs of which its light subunits, i.e., the L-type AA transporters SLC7A5 (LAT1) and SLC7A8 (LAT2) share 48% sequence identity, are not glycosylated, and are composed of 12 TMs with cytoplasmic N-and C-termini [3,16]. The supramolecular organization of these HATs was unraveled by negative stain-and cryo-electron microscopy, i.e., 4F2hc-LAT1 [14,15,17] and 4F2hc-LAT2 [10,16,[18][19][20].…”

Molecular basis for redox control by the human cystine/glutamate antiporter System xc-

“…Recent high resolution cryogenic electron microscopy (cryo-EM) structurers of L-type amino acid transporters LAT1/4F2hc, LAT2/4F2hc and b 0,+ AT/rBAT revealed the structural organisation of the heterodimer [26][27][28][29][30][31] , with the large glycosylated ectodomain sitting atop the transporter. The LAT subunits are members of the amino acid polyamine cation (APC) superfamily 32 , which exhibit a conserved '5+5 inverted topology' fold, wherein the first five TM helices are related to the second five helices via a pseudo two-fold symmetry axis running parallel to the plane of the membrane 33 .…”