Fig. 1.

EBP50 interacts with ezrin cotranslationally. (A) Homology model of closed conformation of human ezrin (Left). Crystal structure showing interaction between C terminus of ezrin with its N-terminal FERM domain (PDB ID code 4RM9) (Center) (10). Homology model of the complex formed between the EBP50 C terminus and the ezrin FERM domain, based on the moesin FERM-EBP50 C-terminal crystal structure (PDB ID code 1SGH) (15) (Right). (B) EBP50 and ezrin interact in cells, but not in vitro. HEK293T cells were cotransfected with His-ezrin and FLAG-EBP50, and proteins detected following immunoprecipitation with anti-FLAG antibody (Left). Purification of His-tagged FL ezrin and ezrin FERM domain from prokaryotic expression system (Center). His-tagged FL ezrin and ezrin FERM domain were immobilized on Ni²⁺-NTA magnetic beads. The interaction with EBP50 was determined using lysates from HEK293T cells overexpressing FLAG-EBP50, followed by elution by heating in SDS-containing sample buffer (Right). (C) Cotranslational interaction between EBP50 and ezrin. Schematic of cotranslational interaction (Left). HEK293T cells were transfected with N-FLAG-EBP50, incubated with or without puromycin (Puro.), and lysates subjected to RIP with IgG or anti-FLAG antibodies followed by qRT-PCR with EZR and PTEN mRNA probes. mRNA was expressed as fold-enrichment compared to isotype-specific IgG RIP-qRT-PCR (Center). Schematic of absent cotranslational interaction in presence of puromycin (Right). Mean + SD, n = 3; **P < 0.01; ns, not significant. (D) Translation is required for EBP50-ezrin interaction. HEK293T cells were transfected with wild type (WT) or start codon mutant (ATG-AAA) FLAG-ezrin construct, and lysates probed with anti-FLAG and anti-GAPDH antibodies (Left). Cells were cotransfected with C-FLAG-ezrin constructs and N-His-EBP50 as in C, and lysates subjected to anti-His RIP-qRT-PCR. mRNA is expressed as fold-enrichment compared to isotype-specific IgG RIP-qRT-PCR (Right). Mean + SD, n = 3; *P < 0.05. (E) Cotranslational interaction of ezrin and EBP50 is unidirectional. Schematic of failed cotranslational interaction of ezrin and nascent EBP50 (Left). HEK293T cells were transfected with C-FLAG-ezrin and subjected to anti-FLAG RIP-qRT-PCR with probes for SLC9A3R1 and MSN mRNA (Right). mRNA is expressed as fold-enrichment compared to isotype-specific IgG RIP-qRT-PCR (Right). Mean + SD, n = 3. (F) Cotranslational interaction of EBP50 with ERM family members. HEK293T cells were transfected with N-FLAG-EBP50 and subjected to anti-FLAG RIP-qRT-PCR with probes for RDX and MSN mRNA. mRNA is expressed as fold-enrichment compared to isotype-specific IgG RIP-qRT-PCR. Mean + SD, n = 3; *P < 0.05, ***P < 0.001.