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