Localization efficiency of EYFP-tagged Pals1 deletion mutants. (A) Schematic representation of the different Pals1-EYFP constructs. Numbers overlying the dotted lines refer to the amino acid positions flanking the internal domain deletions. (B) Localization of wild-type Pals1 and Pals1 deletion mutants in MDCK cells. Cells expressing the various constructs shown in A were fixed and directly visualized. (C) Lysates derived from HEK293 cells expressing the wild-type Pals1-EYFP and the various mutant constructs were resolved by SDS-PAGE. The chimeric protein in which the L27N domain of Pals2 was replaced by the Pals1 L27N domain is denoted as PALS2_PALS1L27N (Fig. 3). The Pals1-EYFP protein harboring a double point mutation in the L27N domain is denoted as PALS1L150GV154G (Fig. 3). (D) Immunostaining of MDCK cells expressing Myc-Pals1 (1–182), which contain only the U1 and L27N domains.

The Pals1 L27N domain binds a protein of ∼200 kD. (A) Lysates prepared from rat brain, mouse brain, MDCK cells, and 293 cells were resolved by SDS-PAGE. Far Western blotting with ³²P-labeled Pals1 L27N probe was performed. (B) Coimmunoprecipitation experiments using antibodies against Myc or mLin-7 and lysates derived from MDCK and 293 cells expressing Myc-Pals1 were performed. Precipitated proteins, resolved by SDS-PAGE, were probed with ³²P-labeled Pals1 L27N domain (top) and immunoblotted with anti-Myc antibody (bottom). The arrowhead points to the IgG heavy chain. (C) The indicated EYFP- and Myc-tagged Pals1 proteins expressed in 293 cells were immunoprecipitated, and bound proteins were resolved by SDS-PAGE. Immunoblotting with anti-EYFP and anti-Myc antibodies confirmed the expression of the Pals constructs (top). Far Western blotting was performed with ³²P-labeled Pals1 L27N probe (bottom). Pals1DM-EYFP refers to EYFP-Pals1 (L150G;V154G), whereas P2P1-EYFP refers to the Pals2_Pals1L27N chimeric protein.

PATJ localizes to tight junctions in MDCK cells. MDCK cells expressing full-length EYFP-PATJ were costained with either anti-Pals1 and anti–ZO-1 antibodies (A) or antiuvomorulin antibody (B) and examined by confocal microscopy.

Full-length CRB1 targets to tight junctions via the interaction between its COOH terminus and Pals1. (A) CRB1 was identified in the critical chromosomal region linked to a severe form of autosomal recessive Retinitis Pigmentosa, RP12. The published domain structure of CRB1 and the protein fragment encoded by the human EST BF912191 are schematically shown. The full-length Myc-CRB1 construct used in this study is also shown. The open rectangle and filled circle represent the signal peptide and the Myc tag, respectively. Rectangles containing horizontal and vertical lines correspond to the EGF-like and C-type lectin domains, respectively. The laminin A G-like domains and the transmembrane region are represented by filled pentagons and the filled rectangle, respectively. (B) Comparison of the transmembrane segments and cytoplasmic tails of Drosophila Crumbs (CRB) and full-length CRB1. Identical residues are in bold. Underlined residues were fused to GST for GST pulldown experiments. (C) MDCK cells stably expressing Myc-CRB1 were immunostained with anti-Myc antibody and antibodies directed against either ZO-1 or Pals1. (D) Immunoprecipitations with preimmune serum (Control IP), anti-PATJ serum, and anti-Myc ascites were performed on lysates derived from the cells in C. Endogenous PATJ and Pals1 were detected by immunoblotting with the respective antisera. CRB1 was detected by blotting with anti-Myc antibody. (E) GST was fused to the COOH-terminal residues of Drosophila Crumbs (GST-CRB) or human CRB1 (GST-CRB1) as indicated in B. 293 lysates containing indicated proteins were incubated with GST-CRB and GST-CRB1 beads. Adsorbed proteins were resolved by SDS-PAGE and detected by immunoblotting with anti-HA and anti-Myc antibodies.

Model depicting the similarities between the mammalian Crumbs–Pals1–PATJ complex and the Drosophila Crumbs–Stardust–Discs Lost complex. In mammalian epithelia, Crumbs interacts with the Pals1 PDZ domain via its COOH terminus. In turn, the L27N domain of Pals1 binds to the NH₂-terminal MRE domain of the multi-PDZ protein, PATJ. In this study, Crumbs, Pals1, and PATJ were all shown to localize to tight junctions. In Drosophila epithelial cells, Crumbs interacts with the Pals1 homologue, CG1617, which encodes a fragment of the Stardust gene. Stardust contains a single L27 domain, which interacts with the MRE domain of the PDZ protein, Discs Lost (DLT). Pals1 and its Drosophila homologue are important adaptors that allow Crumbs to interact with PATJ/Disc Lost.

Targeting of Pals1 in MDCK cells. (A) Pals1 has multiple protein–protein interaction domains including two Lin-2/Lin-7 (L27N and L27C) domains, a PDZ domain, SH3 domain, protein 4.1 binding (4.1B) domain, and GUK domain. (B) MDCK cells were immunostained as described in Materials and methods. The square panels represent digital photomicrographs (x-y dimension of the z-series), and the rectangular panels show the x-z dimension (z-section). (C) Rat kidney sections were immunostained as described in Materials and methods.

The L27N domain of Pals1 targets proteins to tight junctions. (A) Schematic diagram of the deletion and point mutant versions of Pals1. Asterisks denote the double point mutation in the L27N domain (L150G;V154G). (B) Examination of the distribution of Pals1 mutants displayed in A via fluorescence microscopy. (C) Z-section images of MDCK cells expressing wild-type Pals1 or L27N domain mutant Pals1 proteins (green). Red staining represents either ZO-1 or E-cadherin as indicated. (D) Schematic diagram of the wild-type Pals2-EYFP protein and the chimeric protein where the L27N domain of Pals2 is replaced by the Pals1 L27N domain (Pals2_Pals1L27N). We also generated a construct (unpublished data) in which we removed the Pals1 L27N domain from Pals2_Pals1L27N chimera and replaced it with the original Pals2 L27N domain (Pals2_Pals2L27N). (E) Z-section images of MDCK cells expressing Pals1, Pals2, Pals2_Pals1L27N, and Pals2_Pals2L27N proteins. Pals proteins are shown in green. Red staining represents either ZO-1 or E-cadherin.

Purification and identification of the 200 kD binding partner of the Pals1 L27N domain. (A) Large scale Myc-Pals1 and Myc-Pals1Δ(U1 + L27N) coimmunoprecipitations were performed. 5% of the purified proteins were resolved by SDS-PAGE and visualized by silver staining. The arrowhead points to the band representing the 200 kD Pals1 binding partner. (B) Structural alignment between MUPP1, PATJ, and Discs Lost (DLT). Percentage amino acid sequence identity are displayed between the respective MRE domains and PDZ domains (circles and rectangles, respectively). (C) Sequence alignment of the conserved MRE domains in PATJ, MUPP1, C. elegans Discs Large-1 (CeDLG1), SAP97, and Discs Lost (DLT). Identical residues seen in at least three of the five MRE domains are in bold. The sequence data for PATJ have been submitted to GenBank (sequence data available from GenBank/EMBL/DDBJ under accession no. AF397170.

PATJ interacts with the L27N domain of Pals1. (A) Full-length HA epitope-tagged PATJ was coexpressed in 293 cells with either Myc-Pals1 or Myc-Pals1 missing the U1 and L27N domains. Anti-Myc immunoprecipitates from both lysates were resolved by SDS-PAGE. Immunoblotting reveals that HA-PATJ coprecipitates only with full-length Myc-Pals1. (B) GST-PATJ pulldown assays were performed using 293 lysates containing overexpressed Myc-Pals1 or Myc-Pals1ΔL27N. Numbers in parentheses denote the amino acids of PATJ fused to GST. (C) Immunoprecipitations using preimmune serum or anti–mLin-7 antibodies, and lysates from the indicated cell lines were performed. Precipitated proteins were resolved by SDS-PAGE and probed with ³²P-labeled GST-PATJ (1–238). (D) MDCK cell pools expressing Myc-PATJ (1–238) were examined by confocal microscopy. Cells were costained with anti-Pals1 and anti-Myc antibodies.

The Drosophila Pals1 homologue binds to both Crumbs and Discs Lost via its PDZ and L27 domains, respectively. (A) Schematic diagrams of the Drosophila Pals1 homologue, CG1617, and CG1617 fragments used in B and C are shown. All of these proteins were Myc-epitope tagged. (B) GST-CRB pulldowns were performed using 293 cell lysates containing the indicated expressed proteins. (C) 293 lysates containing the denoted Myc-tagged CG1617 proteins were incubated with either GST or GST fused to the NH₂-terminal 135 residues of Discs Lost (DLT), which encompasses the MRE domain. In B and C, bound proteins were resolved by SDS-PAGE and visualized by anti-Myc immunoblot.