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Ubiquitination regulates urea transporter UT‐A1 cell membrane expression
Author(s) -
Chen Guangping,
Fröhlich Otto Otto,
Mistry Abinash C,
Yang Yuan,
Klein Janet D,
Price S. Russ,
Sands Jeff M
Publication year - 2006
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.20.5.a1218-c
Subject(s) - mg132 , cycloheximide , ubiquitin , leupeptin , protein degradation , proteasome inhibitor , microbiology and biotechnology , proteasome , chemistry , intracellular , biology , protease , biochemistry , protein biosynthesis , gene , enzyme
The ubiquitin (Ub)‐proteasome pathway, which selectively degrades intracellular proteins, plays an important role in diverse cellular functions. To identify candidate UT‐A1 binding proteins, we used a 48‐aa fragment of the intracellular C‐terminus of UT‐A1 as bait in a yeast two‐hybrid screen. One positive clone encoded ubiquitin specific protease 24 (USP24), suggesting that ubiquitination may serve as a regulatory marker for degradation. To test this hypothesis, we performed a Ub pull‐down assay and demonstrated ubiquitinated UT‐A1 in UT‐A1‐MDCK cells. Inhibition of the Ub‐proteasome pathway by MG132 dose and time‐dependently increased total UT‐A1 protein abundance. Next, we treated UT‐A1‐MDCK cells with 100 μg/ml cycloheximide (CHX) to block protein synthesis and measured UT‐A1 degradation. The UT‐A1 half‐life is about 10–12 hours. When UT‐A1‐MDCK cells were treated with CHX and 10 μM MG132, the half‐life of UT‐A1 increased. Both cell surface biotinylation and imunostaining showed that cells treated with MG132 increased UT‐A1 cell surface expression. Transepithelial urea flux experiments demonstrated that the cells with increased cell membrane UT‐A1 by MG132 treatment have significantly increased urea transport activity. Transfection of UT‐A1‐MDCK cells with exogenous Ub (pCW7) greatly reduced UT‐A1 abundance in the cell surface. In contrast, lysosomal pathway inhibitors (10 mM methylamine or 50 μg/ml chloroquine) did not affect UT‐A1 abundance. Our studies demonstrate, for the first time, that UT‐A1 is a ubiquitinated protein and is degraded by the proteasome pathway. These findings suggest that ubiquitination plays an important role in the regulation of UT‐A1 bioactivity.