Tyrosine-based Membrane Protein Sorting Signals Are Differentially Interpreted by Polarized Madin-Darby Canine Kidney and LLC-PK1 Epithelial Cells

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 ␤ subunit, which contains a tyrosine-based motif in its cytoplasmic tail, was expressed in MDCK and LLC-PK 1 cells. This protein was restricted to the basolateral membrane in MDCK cells, but was localized to the apical membrane in LLC-PK 1 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-PK 1 cells. A chimera in which the cytoplasmic tail of the H,K-ATPase ␤ subunit protein was replaced with the analogous region of the Na,K-ATPase ␤ subunit polypeptide was localized to both surface domains of MDCK cells. Mutation of tyrosine-20 of the H,K-ATPase ␤ 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-PK 1 cells. The FcRII-B2 protein bears a dileucine motif and is found at the basolateral membrane of both MDCK and LLC-PK 1 cells. These results demonstrate that polarized epithelia are able to discriminate between different classes of specifically defined membrane protein sorting signals.

The Cytoplasmic Tail of Large Conductance, Voltage- and Ca2+-activated K+ (MaxiK) Channel Is Necessary for Its Cell Surface Expression

“…Therefore, we asked whether the problem arises from a failure of assembly or one of cell surface delivery. In addition, because complex renal function relies upon the correct sorting, targeting, and restriction of ion channel proteins to specific regions of the nephronal epithelial cells (43,44), it is important to know the detailed intracellular distribution of renal MaxiK channels at the protein level.…”

The epithelial polarity program: machineries involved and their hijacking by cancer

“…AP1B differs from AP1A (formerly called AP1) in that it possesses an epithelial-specific medium subunit (m1B) instead of the ubiquitous m1A. m1B is expressed by most epithelia but is lacking in LLC-PK1 cells, a kidney cell line believed to derive from proximal convoluted tubule that mislocalizes basolateral proteins to the apical surface (Roush et al, 1998). Transfection of m1B into LLC-PK1 cells promotes normal localization of the missorted basolateral proteins (Folsch et al, 1999(Folsch et al, , 2001Gan et al, 2002).…”

Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter

“…To determine if hUAT is targeted to plasma membranes in living cells, the localization of hUAT/EGFP chimeric proteins has been assessed in a stably transfected immortalized renal epithelial cell line, LLC-PK1, which is known to polarize in culture (40). As demonstrated in sagittal (Figure 3a) and horizontal (Figure 3b) confocal images, hUAT is targeted to both apical and basolateral plasma membranes.…”