Heterodimeric amino acid transporters represent a unique class of transport systems that consist of a light chain that serves as the ‘transporter proper’ and a heavy chain that is necessary for targeting the complex to the plasma membrane. The currently prevailing paradigm assigns no role for the light chains in the cellular processing of these transporters. In this issue of the Biochemical Journal, Sakamoto et al. provide evidence contrary to this paradigm. Their studies with the rBAT –b0,+AT (related to b0,+ amino acid transporter–b0,+-type amino acid transporter) heterodimeric amino acid transporter show that the C-terminus of the light chain b0,+AT contains a sequence motif that serves as the traffic signal for the transfer of the heterodimeric complex from the endoplasmic reticulum to the Golgi. This is a novel function for the light chain in addition to its already established role as the subunit responsible for the transport activity. These new findings also seem to be applicable to other heterodimeric amino acid transporters as well.
The ER (endoplasmic reticulum) is the site of synthesis and assembly of transport proteins that are destined for the plasma membrane . In the case of glycosylated transport proteins, the ER is also the site where glycosylation begins, resulting in asparagine-linked high-mannose-type carbohydrate backbones . If the transporters are multimeric, whether consisting of homomeric or heteromeric complexes, assembly of such multimers occurs in the ER. Only properly assembled transporters are allowed to exit from the ER and enter the Golgi apparatus, where glycosylation is brought into a mature form with further modification of ER-initiated glycosylation . A surveillance mechanism operates as a quality-control system to ensure that only properly assembled proteins are transferred from the ER to the Golgi. If the assembly is incorrect, the resultant proteins are retained in the ER and frequently targeted for ER-associated degradation. This surveillance system is at least partly dependent on specific amino acid sequence motifs in the proteins that are necessary for interaction with chaperones for subsequent escort from the ER to the Golgi. Some of these motifs have been identified for a variety of plasma-membrane proteins [4,5]. In this issue of the Biochemical Journal, Sakamoto et al.  report, for the first time, on the identification of a specific amino acid sequence motif for heterodimeric amino acid transporters to transfer from the ER to the Golgi.
Although most amino acid transporters consist of single proteins, working either as monomers or homomeric multimers, heterodimeric amino acid transporters represent a unique class that consists of two different proteins, namely a heavy chain and a light chain. Heavy chains are type II membrane glycoproteins with a single transmembrane domain. Two such heavy chains have been identified, namely rBAT (related to b0,+ amino acid transporter) or SLC3A1 and 4F2hc or SLC3A2 . There are several light chains, one each for a specific amino acid transporter . The light chains belong to the SLC7 (solute carrier family 7) gene family, possess 12 putative transmembrane domains and represent the catalytic subunit responsible for transport activity. These light chains are not glycosylated. The heavy chains complex with appropriate light chains via a disulfide linkage and target the heterodimeric complexes to the plasma membrane. The primary focus of the paper by Sakamoto et al.  is the amino acid transport system b0,+. This is a heterodimeric amino acid transporter, consisting of rBAT as the heavy chain and b0,+AT as the light chain. It is expressed primarily in the apical membrane of the intestinal and renal absorptive epithelial cells and promotes the absorption of cystine and dibasic amino acids such as lysine, arginine and ornithine. Loss-of-function mutations in either the heavy chain or the light chain lead to cystinuria, owing to inability of the kidneys to reabsorb cystine. Even though the work by Sakamoto et al.  has led to the identification of a specific amino acid sequence motif in the light chain (b0,+AT) that is necessary for the exit of the b0,+AT–rBAT heterodimeric complex from the ER to the Golgi, apparently these studies were not initiated with the goal of identifying such a motif. These investigators have previously reported on a mutation (Pro482→Leu482) in the C-terminal tail of b0,+AT in some patients with cystinuria . This mutation caused loss-of-function of the amino acid transport system, even though there was no impairment in the formation of the heterodimeric complex, glycosylation of the heavy chain rBAT or targeting of the complex to the plasma membrane. The present investigation by Sakamoto et al.  was undertaken to examine the role of the C-terminal tail of b0,+AT in the functional expression of the transporter in greater detail. While they were analysing the transport function, glycosylation pattern of rBAT and the cellular location of the heterodimeric complex using a series of C-terminus deletants and mutants, their investigation led to the identification of a specific amino acid sequence motif that is necessary for the complex to exit from the ER to the Golgi. This motif is Val-Pro-Pro, which is located at position 480–482 in the C-terminal tail of the light chain b0,+AT. When this motif is deleted, b0,+AT is still able to form a complex with the heavy chain, rBAT, but the heterodimeric complex is unable to exit from the ER to the Golgi, as evidenced from the complete loss of transport activity, absence of the complex in the plasma membrane, co-localization of the complex with ER markers and a characteristic glycosylation pattern of rBAT. As indicated above, if the complex is able to travel through the ER and the Golgi, rBAT will have a mature glycosylation pattern. On the other hand, if the complex is unable to exit from the ER to the Golgi, rBAT will have an immature glycosylation pattern, with evidence of ER-dependent glycosylation, but with no evidence of Golgi-dependent additional modification in the glycosylation pattern. Specific glycosidases can be used to distinguish between the immature high-mannose-type carbohydrate moiety that is characteristic of ER-dependent glycosylation and the mature carbohydrate moiety that is characteristic of complete glycosylation with participation of both the ER and the Golgi. Endo H (endoglycosidase H) specifically recognizes the immature high-mannose-type glycosylation; therefore the carbohydrate moiety of rBAT will be sensitive to this glycosidase only if rBAT is unable to exit from the ER to the Golgi and, hence, resides in the ER with no Golgi-dependent modification of its ER-initiated glycosylation. By contrast, rBAT with fully mature glycosylation will be insensitive to Endo H digestion, indicative of its normal passage through the ER and the Golgi. Sakamoto et al.  used this strategy to demonstrate the retention of the b0,+AT–rBAT complex resulting from the deletion of the Val480-Pro481-Pro482 motif in the C-terminal tail of b0,+AT. The insensitivity to Endo H of heterodimeric complexes with such deletants, coupled with co-localization of the complex with ER markers, absence of the complex in the plasma membrane and lack of transport activity, provides compelling evidence to support the claim that the Val480-Pro481-Pro482 motif is the traffic signal for the heterodimeric complex to transfer from the ER to the Golgi.
The identification of the amino acid sequence motif in the light chain b0,+AT as a deciding signal for the transfer of the heterodimeric complex b0,+AT–rBAT from the ER to the Golgi represents a paradigm shift in the field of heterodimeric amino acid transporters. The currently prevailing paradigm in the biogenesis of these amino acid transporters suggests that the heavy chains rBAT and 4F2hc are responsible for the proper trafficking of the heterodimeric complexes . According to this paradigm, the heterodimeric complexes will be recruited to the plasma membrane as long as there are no changes in the heavy chains and dimerization between the heavy chains and the light chains occurs normally. The studies by Sakamoto et al.  reported in this issue of the Biochemical Journal necessitate significant revisions in this paradigm. These studies show that b0,+AT mutants with deletion of the Val480-Pro481-Pro482 motif do dimerize with rBAT properly, as evidenced from the immunoprecipitation experiments. With no changes in rBAT and with the formation of the heterodimeric complex being normal, the current paradigm would predict normal trafficking to the plasma membrane of the complex consisting of the b0,+AT deletant. However, the studies by Sakamoto et al.  show that this is not the case. The amino acid sequence motif plays an obligatory role in the trafficking process. These studies, however, do not eliminate the essential role of the heavy chain, because the motif serves as the traffic signal for the exit from the ER to the Golgi only for the heterodimeric complex, but not for the light chain alone. The ‘old’ paradigm has been described elegantly by Palacin and Kanai , and the new data provided by Sakamoto et al.  dictate modifications in the paradigm as follows: (i) the light chain and the heavy chain are inserted in the ER membrane independently; (ii) the active conformation of the light chain in the membrane does not require the heavy chain; (iii) the light chain and the heavy chain dimerize to form the heterodimeric complex in the ER membrane, and the heavy chain goes through the ER-dependent immature high-mannose-type and Endo H-sensitive glycosylation; (iv) the newly identified signal in the C-terminal tail of the light chain allows the exit of the heterodimeric complex from the ER to the Golgi; (v) the glycosylation of the heavy chain is further modified in the Golgi to generate a mature and Endo H-insensitive pattern; and (vi) the fully processed complex is finally recruited to the plasma membrane in a transport-competent form. This revised paradigm incorporates a novel and essential role of the light chain in the maturation and trafficking of the heterodimeric complex, besides being the catalytic subunit as the ‘transporter proper.’ The Val480-Pro481-Pro482 motif is well conserved in the C-termini of the light chains of other heterodimeric amino acid transporters. Unpublished work (cited in ) from the laboratory of Yoshikatsu Kanai (the senior author of ) suggests that the motif plays a similar role in other transporters as well. Therefore the revised paradigm described here may be applicable not only to b0,+AT–rBAT, but also to other heterodimeric amino acid transporters.
The suggested function for the amino acid sequence motif in the C-termini of the light chains of the heterodimeric amino acid transporters as the ER-to-Golgi traffic signal may only be the tip of the iceberg. Previous studies from the same group of investigators have shown that partial changes in the Val480-Pro481-Pro482 motif do not affect its traffic signal function . Such changes do allow proper maturation, trafficking and glycosylation of the heterodimeric complex and its proper recruitment to the plasma membrane, but the resultant complex in the plasma membrane exhibits no transport activity. This is surprising, because the amino acid sequence motif is present almost at the end of the C-terminal tail and is predicted to be in the cytoplasm. How can mutations in this region influence the transport function? Sakamoto et al.  hypothesize that this region may be involved in some unknown protein–protein interactions. This is logical, but not unprecedented. Protein–protein interactions have been demonstrated to play a role in the modification of transport function in various other transporters. Identification of proteins that participate in protein–protein interaction in heterodimeric amino acid transporters with consequent alterations in transport function will certainly be the next important development in the area of these novel transporters. There have been reports in the literature on some cystinuria patients in whom no mutations have been identified in rBAT or b0,+AT [9,10]. It is possible that mutations in such interacting proteins might be responsible for such cases.