The ammonium transporter RhBG: requirement of a tyrosine-based signal and ankyrin-G for basolateral targeting and membrane anchorage in polarized kidney epithelial cells.
RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations. (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells.
Lopez
Claude
C
INSERM, U665, Institut National de la Transfusion Sanguine, Paris F-75015, France.
Métral
Sylvain
S
Eladari
Dominique
D
Drevensek
Stéphanie
S
Gane
Pierre
P
Chambrey
Régine
R
Bennett
Vann
V
Cartron
Jean-Pierre
JP
Le Van Kim
Caroline
C
Colin
Yves
Y
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000737:chemistry / Q000502:physiology*; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology*; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000378:metabolism; D005822:Genetic Vectors; D006023:Glycoproteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000502:physiology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000737:chemistry*
C490613:ANK3 protein, human; D017487:Ankyrins; D017931:DNA Primers; D018076:DNA, Complementary; D005656:Fungal Proteins; D006023:Glycoproteins; D026901:Membrane Transport Proteins; C422995:RHBG protein, human; D014443:Tyrosine