A novel role of the C-terminus of b0,+AT in the ER–Golgi trafficking of the rBAT–b0,+AT heterodimeric amino acid transporter

Abstract

The heterodimeric complex composed of rBAT (related to b(0,+) amino acid transporter), a single-membrane-spanning glycosylated heavy chain, and b(0,+)AT, a putative 12-membrane-spanning non-glycosylated light chain, is an amino acid transporter that mediates the activity of system b(0,+), a major apical transport system for cystine and dibasic amino acids in renal proximal tubule and small intestine. The C-terminus of b(0,+)AT has been proposed to play an important role in the functional expression of the heterodimeric transporters. In the present study, to reveal the roles of the C-terminus, we analysed b(0,+)AT mutants whose C-termini were sequentially deleted or replaced by site-directed mutagenesis in polarized MDCKII (Madin-Darby canine kidney II), non-polarized HEK-293 (human embryonic kidney-293) and HeLa cells. Although the deletion of C-terminus of b(0,+)AT did not affect the formation of a heterodimer with rBAT, it resulted in the loss of apparent transport function, owing to the failure of the plasma-membrane targeting of rBAT-b(0,+)AT heterodimeric complex associated with incomplete glycosylation of rBAT. A motif-like sequence Val(480)-Pro(481)-Pro(482) was identified in the C-terminus of b(0,+)AT to be responsible for the C-terminus action in promoting the trafficking of rBAT-b(0,+)AT heterodimeric complex from the ER (endoplasmic reticulum) to Golgi apparatus. This is, to our knowledge, the first demonstration of the active contribution of the C-terminus of a light-chain subunit to the intracellular trafficking of heterodimeric transporters. Because the motif-like sequence Val(480)-Pro(481)-Pro(482) is well conserved among the C-termini of light-chain subunits, common regulatory mechanisms could be proposed among heterodimeric amino acid transporters.

Novel cystine transporter in renal proximal tubule identified as a missing partner of cystinuria-related plasma membrane protein rBAT/SLC3A1

“…One of the known functions of heavy chains of HATs is to localize the light chains to the plasma membrane (19,(23)(24)(25)(26). To examine the role of rBAT in the localization of AGT1, AGT1 was expressed with rBAT in HEK293 cells.…”

Collectrin and ACE2 in renal and intestinal amino acid transport

“…The deletion or mutation of the C-terminal signal of b 0,+ AT (Slc7a9) does not prevent complex formation, but leads to the formation of an inactive complex. 45 Interestingly, mutations in the gene encoding the heavy chain or accessory protein rBAT (Slc3a1) as well as in its interacting light chain b 0,+ AT (Slc7a9) are associated with the autosomal disease cystinuria. Mutations on the gene encoding 4F2hc (Slc3a2), which associates with several members of the Slc7 family, are not connected to any physiological anomaly.…”

A case of cystinuria presenting with cerebellar ataxia and dementia

“…Also, there were 25 cases with the P482L homozygous mutation in SLC7A9 and six cases had heterozygous P482L mutations, as in this case. They suggested that the C-terminus of b 0,+ AT/BAT1, where P482L mutation is located, is important both for glycosylating the rBAT subunit and for promoting the trafficking of the cystine transporter from the endoplasmic reticulum to the Golgi apparatus 1 5. However, in the present case we do not yet know if the P482L missense mutation, which intensively suppresses functions of the cystine transporter rBAT–BAT1, alone caused the cystinuria, even in heterozygous form, or whether the second novel mutation in either SLC3A1 or SLC7A9 affected the clinical phenotype.…”