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Biosynthetic sorting of the sodium pump: Visualization of the segregation of newly synthesized epithelial Na,K‐ATPase from apically directed proteins
Author(s) -
Stoops Emily,
Farr Glen,
Caplan Michael J
Publication year - 2012
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.26.1_supplement.885.6
Subject(s) - chemistry , microbiology and biotechnology , apical membrane , endocytosis , transport protein , atpase , golgi apparatus , biophysics , biochemistry , membrane , cell , biology , enzyme
Regulation of transport protein function involves many mechanisms, including endocytosis, recycling and biosynthetic sorting. The Na,K‐ATPase generates the ion gradients that drive most transepithelial transport. In almost every polarized epithelial cell type, the Na,K‐ATPase is restricted to the basolateral domain of the plasma membrane. To study the regulation of Na,K‐ATPase trafficking and its separation from apical cargo, we have created a stable MDCK cell line expressing a SNAP‐tagged version of Na,K‐ATPase and a CLIP‐tagged version of gp135, a glycoprotein that traffics to the apical membrane. The SNAP and CLIP tags are modified forms of a DNA repair protein that react with benzylguanine (SNAP) and benzylcytosine (CLIP) substrates, resulting in covalent linkage of the benzyl group to an active thiol residue within the tag. Using a block/pulse method and SNAP/CLIP‐specific blocking reagents, newly synthesized cohorts of SNAP‐ and CLIP‐tagged protein can be visualized by confocal and electron microscopy, or subjected to biochemical analysis to reveal time‐dependent protein‐protein interactions. Our data suggest that Na,K‐ATPase and gp135 pursue the same post‐synthetic trafficking pathway through the trans‐Golgi network, after which they appear to segregate from one another. Kinetic analysis reveals that Na,K‐ATPase traffics through the biosynthetic pathway more rapidly than the apical gp135.