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. 2005 Jun 1;388(Pt 2):435-43.
doi: 10.1042/BJ20050021.

Heterodimeric amino acid transporter glycoprotein domains determining functional subunit association

Raffaella Franca  1 , Emilija Veljkovic, Stefan Walter, Carsten A Wagner, François Verrey
Affiliations

Heterodimeric amino acid transporter glycoprotein domains determining functional subunit association

Raffaella Franca et al. Biochem J. .

Abstract

The heteromeric amino acid transporter glycoprotein subunits rBAT and 4F2hc (heavy chains) form, with different catalytic subunits (light chains), functional heterodimers that are covalently stabilized by a disulphide bridge. Whereas rBAT associates with b(0,+)AT to form the cystine and cationic amino acid transporter defective in cystinuria, 4F2hc associates with other homologous light chains, for instance with LAT1 to form a system L neutral amino acid transporter. To identify within the heavy chains the domain(s) involved in recognition of and functional interaction with partner light chains, chimaeric and truncated forms of rBAT and 4F2hc were co-expressed in Xenopus laevis oocytes with b(0,+)AT or LAT1. Heavy chain-light chain association was analysed by co-immunoprecipitation, and transport function was tested by tracer uptake experiments. The results indicate that the cytoplasmic tail and transmembrane domain of rBAT together play a dominant role in selective functional interaction with b(0,+)AT, whereas the extracellular domain of rBAT appears to facilitate specifically L-cystine uptake. For 4F2hc, functional interaction with LAT1 was mediated by the N-terminal part, comprising cytoplasmic tail, transmembrane segment and neck, even in the absence of the extracellular domain. Alternatively, functional association with LAT1 was also supported by the extracellular part of 4F2hc comprising neck and glycosidase-like domain linked to the complementary part of rBAT. In conclusion, the cytoplasmic tail and the transmembrane segment together play a determinant role for the functional interaction of rBAT with b(0,+)AT, whereas either cytoplasmic or extracellular glycosidase-like domains are dispensable for the functional interaction of 4F2hc with LAT1.

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Figures

Figure 1
Figure 1. Schematic drawing of heterodimeric amino acid transporter glycoprotein subunits (heavy chains) constructs and summarized results
The domains derived from 4F2hc and rBAT are represented as white and filled rectangles respectively. The summary shows that the intracellular and transmembrane domains of the glycoprotein subunits play a central role in determining the selectivity of the functional association with light chain subunits. TM, transmembrane domain; IP, immunoprecipitation; nd, not determined; 1, immunoprecipitation with antibody against b0,+AT 2, low expression level/stability.
Figure 2
Figure 2. N-glycosylation pattern of rBAT and the chimaeras B44B and BBB4
Deglycosylation reactions with endoglycosidase H (Endo-H) and N-glycosidase F (Endo-F) were performed on lysates of oocytes expressing rBAT or the indicated chimaera and that were biosynthetically labelled with L-[35S]methionine. Immunoprecipitation was with anti-rBAT antibody. hc, heavy chain; Bt, terminally glycosylated rBAT; Bc, core-glycosylated rBAT; Bd, deglyco-sylated rBAT; 4t, terminally glycosylated 4F2hc; 4c, core-glycosylated 4F2hc; 4d, deglycosylated 4F2hc. Molecular mass markers (kDa) are shown on the left (in this and subsequent figures).
Figure 3
Figure 3. Co-immunoprecipitation and cell-surface expression of light-chain subunit b0,+AT and glycoprotein subunit constructs
Oocytes were injected with the indicated cRNAs and, for (A, C, D), the newly synthesized proteins biosynthetically labelled with L-[35S]methionine. (A, C) Immunoprecipitations of the biosynthetically labelled material using an antibody raised against the N-terminus of rBAT. (B) Western blot of cell-surface proteins with an antibody raised against the light chain b0,+AT. (D) Immunoprecipitation of biosyntethically labelled material using the anti-b0,+AT antibody. SDS/PAGE was performed under reducing conditions where indicated (βME; =2-ME). hc, heavy chain; Bt, rBAT terminally glycosylated; 4t, 4F2hc terminally glycosylated; Bc, rBAT core glycosylated; 4c, 4F2hc core glycosylated; B, rBAT; hd117, heterodimer of rBATΔ117 and b0,+AT; B117, rBATΔ117.
Figure 4
Figure 4. b0,+-like transport activity induced by heavy-chain constructs with endogenous oocyte light chain
Oocytes were injected with 10 ng of each cRNA, with the exception of rBATΔ117 in (A) where 30 ng was used. After 2–3 days of expression, uptake (10 min in Na+-free solution) of (A) L-[3H]arginine (100 μM) or (B) L-[14C]cystine (50 μM, in the presence of 10 mM diamide) were measured. When compared with the L-arginine transport rate induced by 4F2hc, that induced by BBB4 and BB4B were significantly higher by ANOVA post-test. For L-cystine, the uptake by none of the chimaeras was statistically different from that with 4F2hc according to ANOVA post-tests, whereas the t test analysis was positive for BBB4, BB44 and BB4B.
Figure 5
Figure 5. Co-immunoprecipitation and cell-surface expression of light-chain subunit LAT1 and glycoprotein subunit constructs
Oocytes were injected with the indicated cRNAs and the newly synthesized proteins biosynthetically labelled with L-[35S]methionine. An antibody against the light chain LAT1 was used for immunoprecipitation. (C) Cell-surface proteins were first labelled with biotin and lanes 2–4 show the immunoprecipitation with anti-LAT1 antibody. This immunoprecipitate was then submitted to streptavindin precipitation to select the cell surface biotinylated proteins (lanes 5–8). SDS/PAGE was performed under reducing conditions where indicated (βME; =2-ME). hc, heavy chain; hd4F2, heterodimer 4F2hc-LAT1; Bt, rBAT terminally glycosylated; 4t, 4F2hc terminally glycosylated; Bc, rBAT core glycosylated; 4c, 4F2hc core glycosylated; hd117 and hd133, heterodimers of LAT1 and 4F2hcΔ117 or 4F2hcΔ133 respectively.
Figure 6
Figure 6. L-type transport activity of LAT1 co-expressed with heavy chain constructs
Oocytes were injected with 10 ng of the indicated cRNAs. After 1 day of expression (4 days for truncations), uptakes of L-[14C]isoleucine (100 μM) were measured (10 min, in Na+-free buffer supplemented with 10 mM L-arginine to block b0,+AT) after injecting 1 nmol of unlabelled L-phenylalanine for trans-stimulation. Transport rates are given as described in the Experimental section. When compared with the L-isoleucine transport rate induced by LAT1 alone, the rate induced by 44B4 and by both truncations, together with LAT1, was significantly higher according to the ANOVA post-test (Bonferroni), whereas that induced by 444B and BB44 reached a level of significance only according to t test analysis.
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