The N-terminal basolateral targeting signal unlikely acts alone in the differential trafficking of membrane transporters in MDCK cells

Biochemistry. 2013 Jul 30;52(30):5103-5116. doi: 10.1021/bi4005914. Epub 2013 Jul 18.

Abstract

We have shown previously, using confocal imaging and transport assays, that the N-terminus of sodium-dependent vitamin C transporter 2 (SVCT2) can redirect apical SVCT1 to the basolateral membrane. Here, the SVCT model was used to further characterize the basolateral targeting peptide signal. Both the length (31 amino acids) and sequence accuracy of the N-terminus of SVCT2 were found to be important in basolateral targeting activity, suggesting a structural requirement. However, the N-terminal basolateral targeting sequence did not appear to act alone, based on analyses of heterologous chimeras. Although diverse N-terminal basolateral targeting signals from multipass membrane proteins can all redirect apical protein from the same gene family to the basolateral membrane, none of the N-terminal basolateral targeting signals can redirect the transmembrane and C-terminal regions from a different gene family. Instead, the presence of these heterologous N-terminal basolateral targeting signals affected the trafficking of otherwise apical protein, causing their accumulation in a stable tubulin-like non-actin structure. Nontargeting N-terminal sequences had no effect. Similar protein retention was observed previously and in this study when the C-terminus of apical or basolateral protein was mutated. These results suggest that the N- and C-termini interact, directly or indirectly, within each gene family for basolateral targeting. Circular dichroism and two-dimensional nuclear magnetic resonance analyses both found a lack of regular secondary structure in the conserved N-terminus of SVCT2, consistent with the presence of partner(s) in the targeting unit. Our finding, a departure from the prevailing single-peptide motif model, is consistent with the evolution of basolateral transporters from the corresponding apical genes. The interaction among the N-terminus, its partner(s), and the cellular basolateral targeting machinery needs to be further elucidated.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Membrane / metabolism*
  • Cell Polarity
  • Conserved Sequence
  • Dogs
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Madin Darby Canine Kidney Cells
  • Models, Biological*
  • Mutant Chimeric Proteins / chemistry
  • Mutant Chimeric Proteins / metabolism
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Protein Sorting Signals*
  • Protein Transport
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Sodium-Coupled Vitamin C Transporters / chemistry
  • Sodium-Coupled Vitamin C Transporters / genetics
  • Sodium-Coupled Vitamin C Transporters / metabolism*

Substances

  • Mutant Chimeric Proteins
  • Mutant Proteins
  • Peptide Fragments
  • Protein Sorting Signals
  • Recombinant Fusion Proteins
  • SLC23A1 protein, human
  • SLC23A2 protein, human
  • Sodium-Coupled Vitamin C Transporters
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins