Sequence- or position-specific mutations in the carboxyl-terminal FL motif of the kidney sodium bicarbonate cotransporter (NBC1) disrupt its basolateral targeting and α-helical structure

Hong C. Li, Joel H. Collier, Ali Shawki, Jai S. Rudra, Emily Y. Li, Bryan MacKenzie, Manoocher Soleimani

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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 α-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 α-helical structure of the C-terminal cytoplasmic domain and for targeting of NBC1 to the basolateral membrane.

Original languageEnglish (US)
Pages (from-to)111-124
Number of pages14
JournalJournal of Membrane Biology
Volume228
Issue number2
DOIs
StatePublished - Mar 2009
Externally publishedYes

Fingerprint

Sodium-Bicarbonate Symporters
Oocytes
Kidney
Mutation
Madin Darby Canine Kidney Cells
Membranes
Protein Disulfide-Isomerases
Peptides
Bicarbonates
Circular Dichroism
Xenopus
Mutagenesis
Epitopes
Cell Membrane
RNA
Amino Acids

Keywords

  • Acid-base transporters
  • Epithelial transport
  • Intestinal and renal transport
  • Intracellular pH regulation

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

Cite this

Sequence- or position-specific mutations in the carboxyl-terminal FL motif of the kidney sodium bicarbonate cotransporter (NBC1) disrupt its basolateral targeting and α-helical structure. / Li, Hong C.; Collier, Joel H.; Shawki, Ali; Rudra, Jai S.; Li, Emily Y.; MacKenzie, Bryan; Soleimani, Manoocher.

In: Journal of Membrane Biology, Vol. 228, No. 2, 03.2009, p. 111-124.

Research output: Contribution to journalArticle

Li, Hong C. ; Collier, Joel H. ; Shawki, Ali ; Rudra, Jai S. ; Li, Emily Y. ; MacKenzie, Bryan ; Soleimani, Manoocher. / Sequence- or position-specific mutations in the carboxyl-terminal FL motif of the kidney sodium bicarbonate cotransporter (NBC1) disrupt its basolateral targeting and α-helical structure. In: Journal of Membrane Biology. 2009 ; Vol. 228, No. 2. pp. 111-124.
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abstract = "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 α-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 α-helical structure of the C-terminal cytoplasmic domain and for targeting of NBC1 to the basolateral membrane.",
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AU - Li, Hong C.

AU - Collier, Joel H.

AU - Shawki, Ali

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AU - Li, Emily Y.

AU - MacKenzie, Bryan

AU - Soleimani, Manoocher

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AB - 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 α-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 α-helical structure of the C-terminal cytoplasmic domain and for targeting of NBC1 to the basolateral membrane.

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KW - Intracellular pH regulation

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