Sequence- or position-specific mutations in the carboxyl-terminal FL motif of the kidney sodium bicarbonate cotransporter (NBC1) disrupt its basolateral targeting and alpha-helical structure.
The sodium-bicarbonate cotransporter NBC1 is targeted exclusively at the basolateral membrane. Mutagenesis of a dihydrophobic FL motif (residues 1013-1014) in the C-terminal domain disrupts the targeting of NBC1. In the present study, we determined the precise constraints of the FL motif required for basolateral targeting of NBC1 by expressing epitope-tagged wild-type and mutant NBC1 in MDCK cells and RNA-injected Xenopus oocytes and examining their subcellular localization. We assayed the functional activity of the mutants by measuring bicarbonate-induced currents in oocytes. Wild-type NBC1 (containing PFLS) was expressed exclusively on the basolateral membrane in MDCK cells. Reversal of the FL motif (PLFS) had no effect on basolateral targeting or activity. Shifting the FL motif one residue upstream (FLPS) resulted in mistargeting of the apical membrane but the FLPS mutant retained its functional activity in oocytes. Shifting the FL motif one residue downstream resulted in a mutant (PSFL) that did not efficiently translocate to the plasma membrane and was instead colocalized with the ER marker, protein disulfide isomerase (PDI). Analysis of circular dichroism (CD) revealed that a short peptide, 20 amino acid residues, of wild-type NBC1 contained a significant alpha-helical structure, whereas peptides in which the FL motif was reversed or C-terminally shifted were disordered. We therefore propose that the specific orientation and the precise location of the FL motif in the primary sequence of NBC1 are strict requirements for the alpha-helical structure of the C-terminal cytoplasmic domain and for targeting of NBC1 to the basolateral membrane.
Li
Hong C
HC
Center on Genetics of Transport, University of Cincinnati, Cincinnati, OH 45267, USA. Hong.Li@uc.edu
Collier
Joel H
JH
Shawki
Ali
A
Rudra
Jai S
JS
Li
Emily Y
EY
Mackenzie
Bryan
B
Soleimani
Manoocher
M
D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002942:Circular Dichroism; D004285:Dogs; D006801:Humans; D018613:Microscopy, Confocal; D009154:Mutation / Q000235:genetics*; D009865:Oocytes; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters