Role of intracellular C‐terminal domain of the catalytic subunit of system b 0,+ for its trafficking and the RACK1 binding.

System b 0,+ , which is defective in cystinuria, is a heteromeric amino acid transporter composed of the catalytic subunit b 0,+ AT/BAT1 light chain ( SLC7A9 ) and the accessory subunit rBAT heavy chain ( SLC3A1 ). Although rBAT has been reported to play a role in apical sorting of this heterodimeric unit, the contribution of b 0,+ AT in the membrane sorting has not been well characterized. Here, we found that b 0,+ AT C‐terminal deletion mutants including a motif‐like sequence “VVPP” failed to transport [ 14 C]L‐cystine in HEK 293 cells expressing rBAT‐b 0,+ AT. When expressed in HeLa cell, b 0,+ AT mutants lacking this motif were not sorted to the apical membrane and retained in ER. We also observed that the deletion of this region interrupted the glycosylation of rBAT. Then, we performed the yeast two‐hybrid screening of human kidney cDNA library using the intracellular part of b 0,+ AT as bait to search for proteins interacting to this region. We found that 15 out of 208 positive clones are scaffolding protein RACK1, a receptor for activated C kinase‐1. siRNA inhibition of RACK1 expression interfered the membrane sorting of this heterodimeric transporter. These results suggested that the b 0,+ AT C‐terminus is required for both the RACK1 binding and the ER‐Golgi transport and, therefore, the full glycosylation of rBAT heavy chain.