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.
Highlights
Amino acid transporters play vital roles to provide cells with important substrates as amino acids and derivatives thereof
Isolated membranes were solubilized in lauryl maltose neopentyl glycol (LMNG)/cholesteryl hemisuccinate (CHS) and bound to nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography resin
The minor fraction of faster migrating bands compared to the covalently linked heterodimeric complex 4F2hc-LAT2 (Figure 1C) were attributed to 4F2hc and LAT2 [20]. We hypothesize that this minor amounts of free 4F2hc and LAT2 arise upon SDS denaturation and/or the SDS-PAGE of the complex, since the second size exclusion chromatography (SEC) elution profile (Figure 1B) displayed only one major peak, which was free from lower molecular
Summary
Amino acid transporters play vital roles to provide cells with important substrates as amino acids and derivatives thereof. Whereas CATs are N-glycosylated, LATs are not Instead, they associate with glycoproteins from the SLC3 family, e.g., 4F2hc (SLC3A2, CD98) or rBAT (SLC3A1), to form heterodimers. They associate with glycoproteins from the SLC3 family, e.g., 4F2hc (SLC3A2, CD98) or rBAT (SLC3A1), to form heterodimers In these complexes, LATs are referred as the light chains and the SLC3 family proteins are referred as the heavy chains. The HAT 4F2hc-LAT2 (SLC3A2-SLC7A8) is an obligatory exchanger It preferably transports neutral as well as small L-amino acids [7,8,9] and the thyroid hormones T3 and T4 [10] in a sodium-independent manner [8]. Whereas the loss of LAT2-dependent transport function is attributed to age-related hearing loss, renal aminoaciduria, and cataract formation [1,13,14,15], an elevated expression level of LAT2 is linked to cancer [16]
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