Abstract
Tyrosine-dependent sequence motifs are implicated in sorting membrane proteins to the basolateral domain of Madin-Darby canine kidney (MDCK) cells. We find that these motifs are interpreted differentially in various polarized epithelial cell types. The H, K-ATPase beta subunit, which contains a tyrosine-based motif in its cytoplasmic tail, was expressed in MDCK and LLC-PK1 cells. This protein was restricted to the basolateral membrane in MDCK cells, but was localized to the apical membrane in LLC-PK1 cells. Similarly, HA-Y543, a construct in which a tyrosine-based motif was introduced into the cytoplasmic tail of influenza hemagglutinin, was sorted to the basolateral membrane of MDCK cells and retained at the apical membrane of LLC-PK1 cells. A chimera in which the cytoplasmic tail of the H,K-ATPase beta subunit protein was replaced with the analogous region of the Na,K-ATPase beta subunit polypeptide was localized to both surface domains of MDCK cells. Mutation of tyrosine-20 of the H,K-ATPase beta subunit cytoplasmic sequence to an alanine was sufficient to disrupt basolateral localization of this polypeptide. In contrast, these constructs all remain localized to the apical membrane in LLC-PK1 cells. The FcRII-B2 protein bears a di-leucine motif and is found at the basolateral membrane of both MDCK and LLC-PK1 cells. These results demonstrate that polarized epithelia are able to discriminate between different classes of specifically defined membrane protein sorting signals.
Highlights
Polarized epithelial cells serve as barriers between compositionally dissimilar environments
Madin-Darby canine kidney (MDCK) cells were stably transfected with the rabbit gastric H,K-ATPase  subunit cDNA
In MDCK cells glycosylphosphatidylinositol-linked proteins are sorted to the apical membrane [36, 37], whereas members of this family are directed to the basolateral domain in Fisher rat thyroid epithelial cells [38]
Summary
A new restriction enzyme site, EagI, was introduced into N28H to provide an overlapping region for annealing the halves of this chimera. 50-l reactions (2 M each primer, 0.1 mM template cDNA, 0.2 mM of each dATP, dTTP, dCTP, dGTP, 1ϫ Vent DNA polymerase reaction buffer, 1 unit Vent polymerase (New England Biolabs, Beverly, MA)) were run under the conditions indicated: N28H; melt; 97 °C, 15 s; anneal; 40 °C, 120 s; extend; 72 °C, 90 s; 30 cycles; dGTP was replaced with a 3:1 ratio of 7-deaza-2Ј-dGTP to dGTP, and H-Y20A; melt; 97 °C 15 s; anneal; 50 °C, 60 s; extend; 72 °C, 60 s; 30 cycles These products were isolated and purified on 1.5% low melting temperature agarose gels (Bio-Rad, Hercules, CA). Contrast and brightness settings were adjusted so that all pixels were in the linear range
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