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

Abstract

Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b 0,+ AT/SLC7A9 and its auxiliary subunit rBAT/ SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b 0,+ AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.amino acid transporter | cystine reabsorption | cystinuria | kidney T he heteromeric amino acid transporter (HAT) family is one of the major amino acid transporter families responsible for cellular uptake and epithelial transport (1-3). HATs form heterodimers composed of a 12 membrane spanning light chain (SLC7) that catalyzes transport functions and a single membrane spanning heavy chain (SLC3) essential for plasma membrane localization and stabilization of the light chains. Two heavy chains, SLC3A1/ rBAT and SLC3A2/4F2hc/CD98hc, covalently bound to light chains via a disulfide bridge have been identified so far (4-6). 4F2hc interacts with most of the light chains in HATs whereas rBAT has been known to form a heterodimer only with b 0,+ AT/ SLC7A9. Because the rBAT-b 0,+ AT complex is presented on the apical membrane of proximal tubules in the kidney and involved in the reabsorption of cystine and dibasic amino acids, the mutations of either rBAT or b 0,+ AT cause cystinuria, a disorder of renal reabsorption of cystine and dibasic amino acids leading to serious renal lithiasis due to low solubility of cystine (7).An unsolved paradox on rBAT and b 0,+ AT has been the discrepancy between the distribution of rBAT and that of b 0,+ AT (5,(8)(9)(10). rBAT is the most abundant in the S3 segmen...

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“…In situ hybridization was performed as described previously (48). A fragment corresponding to nucleotide positions 1620 to 2132 of mouse LAT1 mRNA (NCBI RefSeq NM_011404.3) was amplified by PCR and subcloned into the pCR4-TOPO cloning vector (Invitrogen).…”

Compensatory Renal Hypertrophy and the Uptake of CysteineS-Conjugates of Hg2+in Isolated S2 Proximal Tubular Segments

“…Therefore, we suggest that the elevated expression of OAT1 and System b 0,þ in hypertrophied segments is probably an important mechanism that leads to enhanced uptake and accumulation of mercuric ions in hypertrophied proximal tubules. Na þ -independent transport of cystine (Nagamori et al, 2016).…”

Cystinuria: A Review of Inheritance Patterns, Diagnosis, Medical Treatment and Prevention of Stones

“…The AGT1 protein is encoded by the SLC7A13 gene, and forms a heterodimer with rBAT to facilitate cystine reabsorption in the S3 part of the proximal tubule [11]. The discovery of AGT1 working alongside rBAT in the S3 section of the proximal tubule may help explain the previously recognised paradox of b 0,AT and rBAT being predominantly expressed in different segments of the proximal tubule.…”

“…The discovery of AGT1 working alongside rBAT in the S3 section of the proximal tubule may help explain the previously recognised paradox of b 0,AT and rBAT being predominantly expressed in different segments of the proximal tubule. It is known that b 0,AT expression is highest in the S1 (proximal) segment of the proximal tubule, whilst rBAT expression is mostly in the S3 (distal) segment [11].…”