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. 2001 Apr 2;153(1):191-206.
doi: 10.1083/jcb.153.1.191.

Identification of EPI64, a TBC/rabGAP domain-containing microvillar protein that binds to the first PDZ domain of EBP50 and E3KARP

D Reczek  1 , A Bretscher
Affiliations

Identification of EPI64, a TBC/rabGAP domain-containing microvillar protein that binds to the first PDZ domain of EBP50 and E3KARP

D Reczek et al. J Cell Biol. .

Abstract

The cortical scaffolding proteins EBP50 (ERM-binding phosphoprotein-50) and E3KARP (NHE3 kinase A regulatory protein) contain two PDZ (PSD-95/DlgA/ZO-1-like) domains followed by a COOH-terminal sequence that binds to active ERM family members. Using affinity chromatography, we identified polypeptides from placental microvilli that bind the PDZ domains of EBP50. Among these are 64- and/or 65-kD differentially phosphorylated polypeptides that bind preferentially to the first PDZ domain of EBP50, as well as to E3KARP, and that we call EPI64 (EBP50-PDZ interactor of 64 kD). The gene for human EPI64 lies on chromosome 22 where nine exons specify a protein of 508 residues that contains a Tre/Bub2/Cdc16 (TBC)/rab GTPase-activating protein (GAP) domain. EPI64 terminates in DTYL, which is necessary for binding to the PDZ domains of EBP50, as a mutant ending in DTYLA no longer interacts. EPI64 colocalizes with EBP50 and ezrin in syncytiotrophoblast and cultured cell microvilli, and this localization in cultured cells is abolished by introduction of the DTYLA mutation. In addition to EPI64, immobilized EBP50 PDZ domains retain several polypeptides from placental microvilli, including an isoform of nadrin, a rhoGAP domain-containing protein implicated in regulating vesicular transport. Nadrin binds EBP50 directly, probably through its COOH-terminal STAL sequence. Thus, EBP50 appears to bind membrane proteins as well as factors potentially involved in regulating membrane traffic.

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Figures

Figure 1
Figure 1
Identification of EBP50 PDZ domain–binding candidates. (A) Summary of GST–EBP50 fusion proteins used for affinity chromatography. The cDNA sequences encoding human EBP50 residues 1–358 (FL), residues 1–97 (P1), residues 1–138 (P1+), residues 138–248 (P2), residues 1–248 (P1 + P2), and residues 241–358 (C) were expressed as soluble recombinant proteins fused to GST. These proteins were purified, immobilized on glutathione–agarose beads, and then used for affinity chromatography. (B) Affinity chromatography. Buffer or total detergent soluble extracts of placental microvilli were mixed with GST–agarose beads or GST–EBP50–agarose beads containing the first PDZ domain (P1 or P1+), the second PDZ domain (P2), or both in tandem (P1 + P2). The beads were then washed extensively in buffer at 0.3 M NaCl, and bound proteins were eluted and resolved on a 6–20% silver-stained gradient SDS gel. (C) One-fourth the amount of the samples shown in B were resolved by SDS-PAGE, transferred to PVDF, and then probed with biotinylated EBP50. Arrowheads and brackets indicate specific 120-kD and 64/65-kD binding candidates, respectively. The mobilities of molecular mass standards in kD are indicated at left. DF, dye front. (D) The heterogeneity of the 64/65-kD candidate is due to phosphorylation. A PDZ domain affinity bead eluate from B that was enriched in the 64/65-kD binding candidate was incubated in the presence (+AP) or absence (−AP) of calf intestinal alkaline phosphatase and then resolved by SDS-PAGE on a 10% gel, transferred to PVDF, and overlaid with biotinyl EBP50 probe as in C.
Figure 2
Figure 2
cDNA sequence and genomic structure of the EPI64 gene. (A) Nucleotide and derived protein sequences of human EPI64 cDNA. The TBC/rabGAP domain is underlined. Intron/exon boundaries are indicated by arrowheads, and the COOH-terminal PDZ–binding motif is boxed. These sequence data are available from GenBank/EMBL/DDBJ under accession number AF331038. (B) Schematic diagram of the intron and exon segments comprising the EPI64 gene. Boxes indicate exons and are numbered sequentially from 5′ to 3′.
Figure 4
Figure 4
The 120-kD human placental protein is an isoform of rat brain nadrin. A schematic diagram showing the domain structure of rat brain nadrin is shown with peptides derived from the sequencing of the human 120-kD protein mapped onto it. Peptides are lettered a–g and have the following sequences: a, TEVLSEDLLQIER; b, MLETCGDAENQLALELSQHEVFVEK; c, LVLDWDSVR; d, LAQTSDVNK; e, KPAPAPPK; f, NRPSVPPPPQPPGVHSAGDSSLTNTAPTASK; g, SIFPEMHSDSASK.
Figure 3
Figure 3
Alignment of EPI64 with related protein sequences. (A) EPI64 has a TBC/rabGAP domain. TBC/rabGAP domains of proteins from diverse species were aligned with EPI64 sequences using the CLUSTAL algorithm of the MEGALIGN software program (DNASTAR). The complete sequences of these proteins are available at the following GenBank/EMBL/DDBJ accession numbers: Homo sapiens BAB14454 (BAB14454), Halocynthia roretzi HrPET-1 (BAA81906), Arabidopsis thaliana PAM1 (AAC33763), H. sapiens rab6 GAP (NP_036329), Mus musculus HBLP1 (NP_061245), Saccharomyces cerevisiae Pim1p (AAB01977), H. sapiens VRP (NP_008994), H. sapiens EVI5 (NP_005656), Drosophila melanogaster RN-tre (AAC48286), Bos taurus Lyncein (CAA76943), M. musculus Tbc1 (AAA85223), D. melanogaster Pollux (AAB02200), and H. sapiens Tre-2 USP6 (NP_004496). Residues matching the consensus sequence are shaded. A well-conserved arginine residue, critical for yeast Gyp1p and Gyp7p GAP activity, is indicated (*). (B) EPI64 demonstrates significant homology to HrPET-1 and is closely related to the unknown human protein BAB14454. An alignment of the full protein sequences of EPI64, HrPET-1, and BAB14454 is shown. The TBC/rabGAP domain is overlined. Sequence identities matching the consensus are shaded.
Figure 5
Figure 5
Expression of recombinant EPI64 and mapping of the EBP50 and E3KARP association sites to the extreme COOH terminus of EPI64. (A) Recombinant EPI64 comigates with the 64-kD species from placental microvilli. Recombinant EPI64 purified from bacterial extracts was resolved by SDS-PAGE adjacent to a sample of EPI64 purified from placental microvilli. The proteins were transferred to PVDF and then detected via an EBP50 PDZ domain blot overlay. (B) Coomassie-stained gel of wild-type (DTYL) and COOH-terminal mutant (DTYLA) GST–EPI64 after purification on glutathione–agarose. A fraction of the samples from B were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with biotinyl EBP50 (C), or biotinyl E3KARP (D). Arrows indicate the migration of the full-length GST–EPI64 fusion proteins. Additional signal for lower molecular mass bands in D result from background seen in control blots.
Figure 6
Figure 6
Tissue and cell distribution of EPI64. 100 μg of total proteins from murine tissues and human placenta and 2 and 10 μg of total proteins from isolated human placental microvilli (left), or 400 μg of total proteins from the indicated cultured human cell lines (right) were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with affinity-purified antibodies to EPI64. The mobilities of molecular mass standards in kD are indicated at left. DF, dye front.
Figure 7
Figure 7
Localization of EPI64 in human placenta. Cryosections of placenta were stained with affinity-purified antibodies to EPI64 (A), EBP50 (D), and ezrin (G) and double-labeled for actin using rhodamine phalloidin (B, E, and H). (C, F, and I) Merged images. EPI64 colocalizes with actin in the microvilli-rich region of the syncytiotrophoblast (arrowheads) in a pattern that is very similar to that seen for EBP50 and ezrin. Bar, 40 μm.
Figure 8
Figure 8
Localization of EPI64 in JEG-3 cells. Human JEG-3 cells transiently transfected with Xpress epitope-tagged EPI64 were fixed and double labeled to detect Xpress-tagged EPI64 (A, D, and G), and EBP50 (B), or ezrin (E), or F-actin (H). (C, F, and I) Merged images. Bar, 10 μm.
Figure 9
Figure 9
Localization of the EPI64 COOH-terminal mutant in JEG-3 cells. Human JEG-3 cells transiently transfected with Xpress epitope-tagged EPI64 COOH-terminal mutant were fixed and double labeled to detect Xpress-tagged mutant EPI64 (A, D, and G), and EBP50 (B), or ezrin (E), or F-actin (H). Bar, 10 μm.
Figure 10
Figure 10
Assembly of protein complexes on the isolated domains of EBP50 and ezrin. Buffer or total detergent-soluble extracts of placental microvilli (MV) were mixed with either GST–agarose beads, agarose beads with GST fused to full-length EBP50 (FL), its two PDZ domains (P1 + P2), or its COOH-terminal half containing the ERM binding site (C), or agarose beads with the ezrin NH2-terminal domain (Ez-N). The beads were then washed, bound proteins eluted and resolved by SDS-PAGE, and the resulting gel was silver stained as described in the legend to Fig. 1 B. The mobilities of molecular mass standards are indicated in kD. The migration of specifically bound polypeptides of determined identity are indicated at right. DF, dye front.
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