Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc

Abstract

Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.CD98hc | 4F2hc ectodomain H eteromeric amino acid transporters (HATs) are composed of two subunits, a heavy (SLC3 family) and a light subunit [SLC7 or L-type amino acid transporter (LAT) family] linked by a conserved disulfide bridge (1). HATs are amino acid exchangers (1), and this transport activity resides in the light subunit (2). The heavy subunit (either 4F2hc or rBAT) is essential for trafficking of the holotransporter to the plasma membrane (3, 4). In mammals, six transporters heterodimerize with 4F2hc, and only one heterodimerizes with rBAT. The rBAT/b 0,+ AT complex is a dimer of heterodimers in which the light subunit is required for proper rBAT folding and stability (5, 6). In contrast, 4F2hc-associated transporters are simple heterodimers (6), and possible stabilizing roles of the two subunits in the biogenesis of the heterodimer have not been described.HATs have major impacts on human health and are involved directly in amino acidurias (cystinuria and lysinuric protein intolerance), tumor cell growth, glioma invasion, Kaposi's sarcomaassociated herpesvirus infection, and cocaine relapse (1). In addition to the role of HATs in amino acid transport, 4F2hc heterodimers mediate β1-and β3-integrin signaling (7).Structural information about HATs is scarce (1). The heavy subunits are type II membrane N-glycoproteins with a single transmembrane domain (TMD), an intracellular N terminus, and a large extracellular C terminus with sequence homology with bacterial α-amylases. Indeed, the atomic structure of the extracellular domain (ED) of human 4F2hc (4F2hc-ED) is similar to that of bacterial glucosidases [i.e., a triose phosphate isomerase barr...

Cultivation strategies to enhance productivity of Pichia pastoris: A review

“…Heteromeric amino acid transporters (HATs) are common to all kingdoms of life, and are composed of two different subunits (heavy and light chains, 4F2hc and LAT2 respectively) linked by a conserved disulphide bridge . The heterodimer 4F2hc/LAT2 was successfully overexpressed in P. pastoris, strain KM71H, including the required posttranscriptional modifications by co-transformation of both subunits (Costa et al, 2013, Rosell et al, 2014. However, the correct (and stoichiometric) linkage of the subunits by the disulphide bridge was affected by culture conditions, as studied systematically in bioreactor cultures (unpublished results).…”

Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC

“…The specific mutation N22A in AdiC is interesting because this amino acid residue is located near the substrate-binding pocket and increases the affinity of AdiC for Arg approximately sixfold (10). In addition to their great value for the understanding of the molecular working mechanism of AdiC and other APC superfamily transporters, high-resolution structures of AdiC are very useful for homology modeling of human SLC7 family members (12); for example, of the large-neutral amino acid transporter-1 (LAT1; SLC7A5) (13) and -2 (LAT2; SLC7A8) (14,15). Because structure determination of human transporters-and eukaryotic membrane proteins in general-still represents a major challenge, Significance Disease-causing bacteria are able to survive the strongly acidic environment of the stomach by activating extreme acid-resistance responses.…”

Amino Acid Transport Associated to Cluster of Differentiation 98 Heavy Chain (CD98hc) Is at the Cross-road of Oxidative Stress and Amino Acid Availability

“…3 Recipient of a Severo Ochoa doctoral fellowship. 4 Recipient of a doctoral fellowship from INSERM Region Provence-Alpes Cote d'Azur/Canceropôle PACA. 5 Both authors share last authorship.…”

“…the catalytic part of the transporter (3,4). CD98hc (aka SLC3A2, 4F2, FRP1), the only ubiquitously expressed heavy subunit of heteromeric amino acid transporters, can bind to any of six light subunits (LAT1, LAT2, xCT, y ϩ LAT1, y ϩ LAT2, and asc1), which confer substrate specificity to the heterodimer, referred to as CD98 (1).…”