Fig. 5. Interaction between BP230 and BP180 involves the conserved Y subdomain of plakins and mapping of the interaction site on BP230 for ß4. The PJ69-4A yeast strain was co-transformed with a pAS2.1- or pPACT2-derived vector encoding BP180^1-401 or ß4^1115-1666 and the corresponding complementary vectors encoding various fragments of BP230, desmoplakin or plectin as indicated. Two-hybrid interactions were analyzed by growth on selective SC-LTHA plates. (A) Top, schematic representation of BP230. The subdomains NN, Z, Y, X, W and V are those described by Green et al. (Green et al., 1992). These subdomains as well as the homologous repeat sequences designated B and C are indicated by boxes, shaded from white to gray. The box representing the rod domain has been shaded more darkly. Bottom, the interaction of BP180^1-401 with the various fragments of BP230 could be shown independently of whether the cDNA constructs were cloned into the pAS2.1 or the pACT2 vector. Because of an autonomous transactivation of pAS2.1-ß4^1115-1666, the BP230 binding activity of ß4^1115-1666 could only be determined with the combination of pACT2-ß4^1115-1666 and pAS2.1-BP230. (B) Interactions of BP180^1-401 with the Z-Y and Y domains of BP230, plectin and desmoplakin (DP) were only revealed when the cDNAs encoding the various domains of BP230, plectin and desmoplakin were inserted into pACT2 and the cDNA for BP180^1-401 was inserted into pAS2.1. (C) The ß-galactosidase activity of the yeast transformants expressing BP180^1-401 and the indicated BP230, plectin and desmoplakin constructs was quantified in a liquid culture assay using O-nitrophenyl ß-D-galactopyranoside as substrate. The negative interaction controls are pAS2.1-BP180^1-401/pACT2 (2.9±0.3 ß-galactosidase units) and pAS2.1/pACT2 (2.4±0.2 ß-galactosidase units) and the positive controls (not shown) are p53/pSV-40 large T (77.3±10.1 ß-galactosidase units) and the complete Gal4 transcription factor in pCL1 (266.8±16.3 ß-galactosidase units). (D) Interactions between BP230 mutants and ß4^1115-1666 were assayed with the BP230 mutants fused to the Gal4 (BD) in pAS2.1 and ß4^1115-1666 fused to the Gal4 (AD) in pACT2. For further details, see Fig. 3. In panel D, the amino acids GGG, GSG and G correspond to the linker sequences placed in between the Gal4 (AD)- and the BP230-specific sequences. In B and D note that the interaction between BP230 and the cytodomain of BP180 only requires the Y domain of BP230, whereas for the interaction with ß4 the 56-most N-terminal residues of BP230 are involved. Panel E shows an alignment of the Z-Y regions in the different plakin proteins.

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