An apical expression signal of the renal type IIc Na+-dependent phosphate cotransporter in renal epithelial cells.
The type IIc Na(+)-dependent phosphate cotransporter (NaPi-IIc) is specifically targeted to, and expressed on, the apical membrane of renal proximal tubular cells and mediates phosphate transport. In the present study, we investigated the signals that determine apical expression of NaPi-IIc with a focus on the role of the N- and the C-terminal tails of mouse NaPi-IIc in renal epithelial cells [opossum kidney (OK) and Madin-Darby canine kidney cells]. Wild-type NaPi-IIc, the cotransporter NaPi-IIa, as well as several IIa-IIc chimeras and deletion mutants, were fused to enhanced green fluorescent protein (EGFP), and their cellular localization was analyzed in polarized renal epithelial cells by confocal microscopy and by cell-surface biotinylation. Fluorescent EGFP-fused NaPi-IIc transporter proteins are correctly expressed in the apical membrane of OK cells. The apical expression of N-terminal deletion mutants (deletion of N-terminal 25, 50, or 69 amino acids) was not affected by truncation. In contrast, C-terminal deletion mutants (deletion of C-terminal 45, 50, or 62 amino acids) did not have correct apical expression. A more detailed mutational analysis indicated that a domain (amino acids WLHSL) in the cytoplasmic C terminus is required for apical expression of NaPi-IIc in renal epithelial cells. We conclude that targeting of NaPi-IIc to the apical cell surface is regulated by a unique amino acid motif in the cytoplasmic C-terminal domain.
Ito
Mikiko
M
Department of Molecular Nutrition, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan.
Sakurai
Aya
A
Hayashi
Keiji
K
Ohi
Akiko
A
Kangawa
Natsumi
N
Nishiyama
Takashi
T
Sugino
Sakiko
S
Uehata
Yoko
Y
Kamahara
Akihiro
A
Sakata
Masae
M
Tatsumi
Sawako
S
Kuwahata
Masashi
M
Taketani
Yutaka
Y
Segawa
Hiroko
H
Miyamoto
Ken-ichi
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D018938:Caco-2 Cells; D016764:Cell Polarity; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D009893:Opossums; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa / Q000235:genetics / Q000378:metabolism; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb / Q000235:genetics / Q000378:metabolism; D050615:Sodium-Phosphate Cotransporter Proteins, Type IIc / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D014162:Transfection
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C493554:SLC34A1 protein, human; C493558:Slc34a2 protein, mouse; C493563:Slc34a3 protein, mouse; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; D050615:Sodium-Phosphate Cotransporter Proteins, Type IIc; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins