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Determinants of AQP6 trafficking to intracellular sites versus the plasma membrane in transfected mammalian cells
Author(s) -
Beitz Eric,
Liu Kun,
Ikeda Masahiro,
Guggino William B.,
Agre Peter,
Yasui Masato
Publication year - 2006
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1042/bc20050025
Subject(s) - intracellular , biology , transfection , green fluorescent protein , aquaporin , cytosol , microbiology and biotechnology , n terminus , vesicle , transport protein , membrane , biochemistry , peptide sequence , gene , enzyme
Background information . Most AQPs (aquaporins) function at the plasma membrane, however AQP6 is exclusively localized to membranes of intracellular vesicles in acid‐secreting type‐A intercalated cells of renal collecting ducts. The intracellular distribution indicates that AQP6 has a function distinct from trans‐epithelial water movement. Results . We show by mutational analyses and immunofluorescence that the N‐terminus of AQP6 is a determinant for its intracellular localization. Presence or absence at the plasma membrane of AQP6 constructs was confirmed by electrophysiological methods. Addition of a GFP (green fluorescent protein) or a HA (haemagglutinin) epitope tag (GFP—AQP6 or HA—AQP6) to the N‐terminus of AQP6, directed AQP6 to the plasma membranes of transfected Madin—Darby canine kidney cells. In contrast, addition of a GFP tag to the C‐terminus (AQP6–GFP) caused the protein to remain intracellular, similar to untagged wild‐type AQP6. Replacement of the N‐terminus of AQP6 by that of AQP1 also directed AQP6 to the plasma membranes, whereas the N‐terminus of AQP6 retained AQP1 in cytosolic sites. Conclusion . Our results suggest that the N‐terminus of AQP6 is critical for trafficking of the protein to the intracellular sites. Moreover, our studies provide an approach for future identification of proteins involved in vesicle sorting in the acid‐secreting type‐A intercalated cells.