Characterization of a di-leucine-based signal in the cytoplasmic tail of the nucleotide-pyrophosphatase NPP1 that mediates basolateral targeting but not endocytosis

Citation
V. Bello et al., Characterization of a di-leucine-based signal in the cytoplasmic tail of the nucleotide-pyrophosphatase NPP1 that mediates basolateral targeting but not endocytosis, MOL BIOL CE, 12(10), 2001, pp. 3004-3015
Citations number
59
Categorie Soggetti
Cell & Developmental Biology
Journal title
MOLECULAR BIOLOGY OF THE CELL
ISSN journal
10591524 → ACNP
Volume
12
Issue
10
Year of publication
2001
Pages
3004 - 3015
Database
ISI
SICI code
1059-1524(200110)12:10<3004:COADSI>2.0.ZU;2-V
Abstract
Enzymes of the nucleotide pyrophosphatase/phosphodiesterase (NPPase) family are expressed at opposite surfaces in polarized epithelial cells. We inves tigated the targeting signal of NPP1, which is exclusively expressed at the basolateral surface. Full-length NPP1 and different constructs and mutants were transfected into the polarized MDCK cell line. Expression of the prot eins was analyzed by confocal microscopy and surface biotinylation. The bas olateral signal of NPP1 was identified as a di-leucine motif located in the cytoplasmic tail. Mutation of either or both leucines largely redirected N PP1 to the apical surface. Furthermore, addition of the conserved sequence AAASLLAP redirected the apical nucleotide pyrophosphatase/phosphodiesterase NPP3 to the basolateral surface. Full-length NPP1 was not significantly in ternalized. However, when the cytoplasmic tail was deleted upstream the di- leucine motif or when the six upstream flanking amino acids were deleted, t he protein was mainly found intracellularly. Endocytosis experiments indica ted that these mutants were endocytosed from the basolateral surface. These results identify the basolateral signal of NPP1 as a short sequence includ ing a di-leucine motif that is dominant over apical determinants and point to the importance of surrounding amino acids in determining whether the sig nal will function as a basolateral signal only or as an endocytotic signal as well.