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Intracellular Loop I is involved in bile acid transporter (SLC10A2) degradation by the proteasome
Author(s) -
Silva Tatiana Claro,
Swaan Peter W.
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.746.1
Subject(s) - intracellular , proteasome , transporter , degradation (telecommunications) , chemistry , microbiology and biotechnology , biochemistry , biology , computer science , gene , telecommunications
The human apical sodium‐dependent bile acid transporter (hASBT, SLC10A2) is the chief intestinal bile acid transporter. Modulation of proteasomal degradation is among ASBT's short‐term adaptive response to potentially cytotoxic bile acid levels, as well as inflammation. Previous findings from our laboratory suggest Gly 50 as possibly involved in ASBT proteasomal degradation. These observations prompted us to further investigate the role of the intracellular loop 1 (IL1) in this process. Here, we performed Ala replacements of IL1 (Cys 51 ‐Phe 72 ) amino acid residues and the mutant transporter proteins were transiently transfected in COS‐1 cells. Mutants G50A, C51A, and N52A showed decreased protein levels, which were rescued by MG132 treatment. G50–N52 are clustered at the TM1/IL1 interface and are flanked by Lysine residues K56, K57, and K63, which are potential targets for ubiquitination. Next, protein synthesis was inhibited with cycloheximide to examine the rate of degradation of select mutants, and showed altered rates for G50A, C51A, N52A, K57A and K63A, compared to the wt‐ASBT. Based on these results, we confirm that residues clustered at the TM1/IL1 interface play a crucial role in ASBT proteasomal degradation, possibly by stabilizing a protein conformation that would prevent IL1 lysine residues from being prematurely exposed to ubiquitin labeling. Supported by NIH RO1 DK61425.