Identification of a carboxyl-terminal motif essential for the targeting of Na+-HCO-3 cotransporter NBC1 to the basolateral membrane.
The Na+-HCO3- cotransporter NBC1 is located exclusively on the basolateral membrane and mediates vectorial transport of bicarbonate in a number of epithelia, including kidney and pancreas. To identify the motifs that direct the targeting of kidney NBC1 to basolateral membrane, wild type and various carboxyl-terminally truncated kidney NBC1 mutants were generated, fused translationally in-frame to GFP, and transiently expressed in kidney epithelial cells. GFP was linked to the NH2 terminus of NBC1, and labeling was examined by confocal microscopy. Full-length (1035 aa) and mutants with the deletion of 3 or 20 amino acids from the COOH-terminal end of NBC1 (lengths 1032 and 1015 aa, respectively) showed strong and exclusive targeting on the basolateral membrane. However, the deletion of 26 amino acid residues from the COOH-terminal end (length 1010 aa) resulted in retargeting of NBC1 to the apical membrane. Expression studies in oocytes demonstrated that the NBC1 mutant with the deletion of 26 amino acid residues from the COOH-terminal end is functional. Additionally, the deletion of the last 23 amino acids or mutation in the conserved residue Phe at position 1013 on the COOH-terminal end demonstrated retargeting to the apical membrane. We propose that a carboxyl-terminal motif with the sequence QQPFLS, which spans amino acid residues 1010-1015, and specifically the amino acid residue Phe (position 1013) are essential for the exclusive targeting of NBC1 to the basolateral membrane.
Li
Hong C
HC
Department of Medicine, University of Cincinnati, Cincinnati, Ohio 45267, USA.
Worrell
Roger T
RT
Matthews
Jeffrey B
JB
Husseinzadeh
Holleh
H
Neumeier
Lisa
L
Petrovic
Snezana
S
Conforti
Laura
L
Soleimani
Manoocher
M
D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004594:Electrophysiology; D017353:Gene Deletion; D005822:Genetic Vectors; D006801:Humans; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018075:RNA, Complementary / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry*; D014162:Transfection; D014981:Xenopus
D018076:DNA, Complementary; D018075:RNA, Complementary; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; D010649:Phenylalanine; D004247:DNA