Premium
Apical membrane targeting of ROMK2 is independent of N‐linked glycosylation
Author(s) -
Collins Valerie Margaret,
Cooper Gordon John,
White Stanley John
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.20.4.a328-b
Subject(s) - glycosylation , apical membrane , fusion protein , n linked glycosylation , green fluorescent protein , microbiology and biotechnology , chemistry , kdel , membrane protein , biology , membrane , biochemistry , glycan , endoplasmic reticulum , glycoprotein , golgi apparatus , gene , recombinant dna
ROMK2, an inwardly rectifying K + channel, is located on the apical membranes of the thick ascending limb (TAL) and distal nephron. Little is known about the mechanisms or motifs that determine apical targeting of the channel. N‐Glycans participate in the surface expression of several membrane proteins (1). ROMK2 contains a single glycosylation consensus sequence (N X S/T) in the first extracellular loop (residues 98–100). Currently, we investigated the role of N‐glycans as a sorting determinant for EGFP‐ROMK2 (WT) fusion proteins in polarized MDCK cells. Filter‐grown MDCK I cells stably expressing WT were treated with 20 μg/ml cycloheximide (CX) and allowed to recover for 3, 6 and 24 hours in the absence or presence of 10 μg/ml tunicamycin (TM) to inhibit glycosylation (n = 3 – 5). Western blotting confirmed the glycosylation state of the EGFP‐fusion proteins. WT was targeted to the apical membrane in untreated MDCK I cells. Treatment with CX reduced protein levels. Fluorescence was not evident at the apical pole. Following removal of CX, fluorescence returned to control levels and fusion protein was targeted to the apical membrane irrespective of the state of glycolsylation. In a second set of experiments, confluent monolayers of MDCK II cells were transiently transfected with EGFP‐ROMK2 in which the glycosylation site was removed by site directed mutagenesis (N98Q). Fluorescence was predominantly located at the apical pole (n = 3 – 5 transfections). These results indicate that ROMK2 is trafficked to the apical membrane independent of glycosylation of the channel.