Role of X interface in the association and dissociation of strand-swap dimers. (A) Cells expressing the parental mutant EcDendra-Δ748-KL (Δ748) and its X dimer mutant K14E (K14E) were chased in media containing 1 µM tunicamycin for 1, 2, or 3 h (indicated above the lanes). In the presence of this inhibitor, cadherin is not glycosylated (arrows) and can be distinguished from the intact form (arrowheads) that had been present in the cells before the addition of tunicamycin. Top blots show total cell lysates (Total Lysates) of these cultures stained with an antibody specific for endogenous cadherin (Ec). Note the gradual accumulation of the unglycosylated form. The same lysates were immunoprecipitated using anti-Dendra2 antibody, and Dendra-tagged cadherin mutants (Dn) and coimmunoprecipitated endogenous cadherin (Ec) were revealed by Western blotting. Note that the X dimer mutation does not delay the formation of strand-swap dimers between the Dendra-tagged mutants and endogenous cadherin. (B) A-431 cells expressing myc-tagged mutant EcMyc-Δ748-KL were co-cultured overnight in low-calcium media with A-431 cells expressing either the same version of the Dendra2-tagged mutant (Δ748) or its K14E mutant (K14E). After different incubation periods in high calcium (indicated in min), cells were lysed, anti-Dendra precipitated, and analyzed for adhesive dimers using anti-myc or, for total (lateral and adhesive dimers), using the endogenous E-cadherin–specific antibody. Note that at the starting point (0), neither cell co-culture has adhesive dimers. High calcium triggers the rapid assembly of adhesive dimers between myc- and Dendra-tagged parental mutants. The X dimer K14E mutant forms adhesive dimers very slowly; cis dimer is a predominant form even after 30 min in high calcium. Note also that at any time, the total amounts of dimers detected by anti-E-cadherin remain at the same level in both co-cultures. Bars in A and B indicate denote the relative positions of 116-kD molecular marker (β-galactosidase). (C) Native gel electrophoresis of total cell lysates (anti-Dendra staining) detects two major (low, arrow; high, arrowhead) molecular weight complexes. Cells were cultured overnight in low calcium (−) and placed to high calcium for 30 min (+). Note, the cells expressing K14E mutant completely lack the low-molecular-weight complex. Bars at the left margin denote the relative positions of molecular markers (molecular weights are indicated).