Determinants of AQP6 trafficking to intracellular sites versus the plasma membrane in transfected mammalian cells.
BACKGROUND
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.


CONCLUSIONS
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.
Beitz
Eric
E
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Liu
Kun
K
Ikeda
Masahiro
M
Guggino
William B
WB
Agre
Peter
P
Yasui
Masato
M
D000595:Amino Acid Sequence; D000818:Animals; D051398:Aquaporin 1 / Q000235:genetics; D051403:Aquaporin 6 / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D049452:Green Fluorescent Proteins / Q000235:genetics; D006388:Hemagglutinins / Q000235:genetics; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D021382:Protein Sorting Signals / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D051403:Aquaporin 6; D006388:Hemagglutinins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D051398:Aquaporin 1; D049452:Green Fluorescent Proteins