The Heavy Chain 4F2hc Modulates the Substrate Affinity and Specificity of the Light Chains LAT1 and LAT2

Abstract

The human L-type amino acid transporters LAT1 and LAT2 mediate the transport of amino acids and amino acid derivatives across plasma membranes in a sodium-independent, obligatory antiport mode. In mammalian cells, LAT1 and LAT2 associate with the type-II membrane N-glycoprotein 4F2hc to form heteromeric amino acid transporters (HATs). The glycosylated ancillary protein 4F2hc is known to be important for successful trafficking of the unglycosylated transporters to the plasma membrane. The heavy (i.e., 4F2hc) and light (i.e., LAT1 and LAT2) chains belong to the solute carrier (SLC) families SLC3 and SLC7, and are covalently linked by a conserved disulfide bridge. Overexpression, absence, or malfunction of certain HATs is associated with human diseases and HATs are therefore considered therapeutic targets. Here, we present a comparative, functional characterization of the HATs 4F2hc-LAT1 and 4F2hc-LAT2, and their light chains LAT1 and LAT2. For this purpose, the HATs and the light chains were expressed in the methylotrophic yeast Pichia pastoris and a radiolabel transport assay was established. Importantly and in contrast to mammalian cells, P. pastoris has proven useful as eukaryotic expression system to successfully express human LAT1 and LAT2 in the plasma membrane without the requirement of co-expressed trafficking chaperone 4F2hc. Our results show a novel function of the heavy chain 4F2hc that impacts transport by modulating the substrate affinity and specificity of corresponding LATs. In addition, the presented data confirm that the light chains LAT1 and LAT2 constitute the substrate-transporting subunits of the HATs, and that light chains are also functional in the absence of the ancillary protein 4F2hc.

Yeast Cell-Based Transport Assay for the Functional Characterization of Human 4F2hc-LAT1 and ‐LAT2, and LAT1 and LAT2 Substrates and Inhibitors

“…Human 4F2hc-LAT1, LAT1, 4F2hc-LAT2 or LAT2 were expressed in the methylotrophic yeast P. pastoris. We showed in previous reports that not only the HATs 4F2hc-LAT1 and -LAT2, but also the light subunits LAT1 and LAT2 in absence of ancillary protein are properly folded, correctly trafficked to the plasma membrane and functional in P. pastoris (Rosell et al, 2014;Kantipudi et al, 2020).…”

“…Towards establishment of a robust assay for ligand screening and functional characterization, we have optimized, applied and validated a previously reported radioligand assay using P. pastoris overexpressing human 4F2hc-LAT1 or -LAT2, and the substrate [ 3 H]L-leucine as radioligand. In contrast to the previously reported HEK293 cell assay (Khunweeraphong et al, 2012), 4F2hc is co-expressed with LAT1 or LAT2 in the Pichia-based assay, thus not limiting 4F2hc availability and boosting expression of HATs (Costa et al, 2013;Rosell et al, 2014;Kantipudi et al, 2020). Interestingly, and in absence of 4F2hc co-expression, Pichia is also able to express functionally the light subunits LAT1 and LAT2 alone (Costa et al, 2013;Rosell et al, 2014;Kantipudi et al, 2020).…”

“…Cloning of the human HATs and LATs into the pPICZB vector (Thermo Fisher Scientific, Waltham, MA, United States), electrotransformation of competent P. pastoris strain KM71H cells (Thermo Fisher Scientific, Waltham, MA, United States) and selection of clones with high protein expression levels was performed as described in detail previously: see for 4F2hc-LAT1 and LAT1 (Kantipudi et al, 2020), and for 4F2hc-LAT2 and LAT2 (Costa et al, 2013). Cell growth and expression conditions of Pichia clones overexpressing human 4F2hc-LAT1, LAT1, 4F2hc-LAT2 or LAT2, and untransformed P. pastoris KM71H cells (control) was conducted according to Kantipudi et al (Kantipudi et al, 2020). Resulting cells were resuspended in transport buffer (150 mM choline chloride, 1 mM MgCl 2 , 1 mM CaCl 2 , 10 mM Tris-HEPES, pH 7.4) containing 50% (v/v) glycerol and the OD 600 was adjusted to 40.…”

“…In contrast to the previously reported HEK293 cell assay (Khunweeraphong et al, 2012), 4F2hc is co-expressed with LAT1 or LAT2 in the Pichia-based assay, thus not limiting 4F2hc availability and boosting expression of HATs (Costa et al, 2013;Rosell et al, 2014;Kantipudi et al, 2020). Interestingly, and in absence of 4F2hc co-expression, Pichia is also able to express functionally the light subunits LAT1 and LAT2 alone (Costa et al, 2013;Rosell et al, 2014;Kantipudi et al, 2020). This allows evaluating possible contributions of the heavy chain 4F2hc on ligand binding and transport inhibition through selected substrates and inhibitors.…”

“…LAT1 (SLC7A5) and LAT2 (SLC7A8) are isoforms of the system L of amino acid transporters requiring the heavy chain 4F2 (4F2hc) for functional expression at the plasma membrane (Kanai et al, 1998;Pineda et al, 1999;Segawa et al, 1999). Furthermore, we recently showed that 4F2hc can modulate the substrate affinity and specificity of the light chains LAT1 and LAT2 (Kantipudi et al, 2020). In addition to these two LAT specific functions, the ancillary protein 4F2hc has multifunctional roles such as in cell adhesion, cell fusion, integrin signaling and regulation of macrophage activation via galectin-3 (Fenczik et al, 1997;Tsurudome and Ito, 2000;Feral et al, 2005;MacKinnon et al, 2008).…”