Abstract
Heteromeric amino acid transporters (HATs) are protein complexes that catalyze the transport of amino acids across plasma membranes. HATs are composed of two subunits, a heavy and a light subunit, which belong to the solute carrier (SLC) families SLC3 and SLC7. The two subunits are linked by a conserved disulfide bridge. Several human diseases are associated with loss of function or overexpression of specific HATs making them drug targets. The human HAT 4F2hc-LAT2 (SLC3A2-SLC7A8) is specific for the transport of large neutral L-amino acids and specific amino acid-related compounds. Human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast Pichia pastoris and pure recombinant protein purified. Here we present the first cryo-electron microscopy (cryo-EM) 3D-map of a HAT, i.e., of the human 4F2hc-LAT2 complex. The structure could be determined at ~13 Å resolution using direct electron detector and Volta phase plate technologies. The 3D-map displays two prominent densities of different sizes. The available X-ray structure of the 4F2hc ectodomain fitted nicely into the smaller density revealing the relative position of 4F2hc with respect to LAT2 and the membrane plane.
Highlights
Amino acids are fundamental nutrients and are involved in numerous cellular processes ranging from energy production to signaling and protein synthesis
According to blue native (BN)-polyacrylamide gel electrophoresis (BN-PAGE), the molecular weight (MW) of the detergent-solubilized, purified complex was estimated at about 230 kDa, whereas the calculated MW of the complex based on the primary amino acid sequences of 4F2hc and LAT2 is about 120 kDa
Since no protein bands below 230 kDa were detected using BN-PAGE and Western blot analysis (Figure 1), the use of lauryl maltose neopentyl glycol (LMNG)/cholesteryl hemisuccinate (CHS) detergent mixture for purification turned out to be beneficial for 4F2hc-LAT2 heterodimer stability
Summary
Amino acids are fundamental nutrients and are involved in numerous cellular processes ranging from energy production to signaling and protein synthesis. The amino acid transporter LAT2 (SLC7A8) preferably transports large neutral L-amino acids and functions as obligatory exchanger [4] It is associated with the heavy chain subunit 4F2hc (CD98), which is a type II membrane N-glycoprotein. 3D-maps at about 20 Å resolution of the 4F2hc-LAT2 complex were obtained using single particle analysis and negative-stain electron microscopy [7,13]. We present the first cryo-EM 3D-map of a HAT, i.e., of the human 4F2hc-LAT2 complex This structure at ~13 Å resolution was successfully obtained using the direct electron detector and VPP technologies. The available X-ray structure of 4F2hc-ED was fitted into the obtained density of human 4F2hc-LAT2 and revealed the relative positions of the heavy subunit with respect to the light subunit and the membrane plane
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