Figure 3.

Intracellular localization of Fez1 protein and characterization of Fez1-interacting proteins. (A and B) Cytoplasmic localization of Fez1 protein. (A) Proteins were extracted from the cytoplasm (C1) and nucleus (N) of 293 cells, electrophoresed, and immunoblotted with anti-Fez1 (lanes 1–3) or anti-tubulin antibody (lanes 4–6) by using 40 μg of protein per lane. C2, intermediate extract. (B) GFP-fusion Fez1 expression vector was transfected into Fez1-negative HeLaS3 cells (II), and GFP expression was observed under a fluorescent microscope. (I) GFP vector control transfectant. (Bar = 5 μm.) (C) In vitro binding of EF1γ to Fez1. The interaction was shown by binding assay with [³⁵S]methionine-labeled in vitro-translated EF1γ protein (lanes 1–8), which was incubated with GST-Fez1 (67 kDa; lane 2) or GST-C-terminal truncated Fez1 (40 kDa, corresponding to nucleotides 1–1128; lanes 4 and 7). Lanes 1, 3, and 6, addition of only GST protein as negative control; lanes 5 and 8, translated EF1γ protein alone as positive control (showing input). Reaction was shown in two different buffers, buffer A (lanes 1–4) and buffer B (lanes 6 and 7), as described in text. Binding of in vitro-translated EF1α (lanes 9 and 10) to GST-Fez1 (lane 10) was analyzed with buffer B. Lane 9, addition of only GST; lane 11, in vitro-translated protein alone, showing input. (D) In vitro binding of Fez1 proteins to N-terminal EF1γ protein. Similar to C, binding assay in buffer B was performed with in vitro-translated full-length 67-kDa Fez1 (lanes 1 and 2) or truncated 40-kDa Fez1 (lanes 3 and 4), incubated with GST-N-terminal EF1γ protein (lanes 2 and 4). Lanes 1 and 3, addition of only GST. In vitro-translated full-length 67-kDa (lane 5) or truncated 40-kDa Fez1 (lane 6) were shown as positive controls. (E) Interaction of Fez1 with EF1γ in transfectants. Full-length FEZ1 or EF1γ cDNA was ligated to pcDNA4V5 or pcDNA3His vector to express V5 tag- or EXP tag-fused protein, respectively. HeLaS3 cells were cotransfected with the vectors. Immunoblot analysis with antitag antibodies shows transfectants express ≈70-kDa Fez1 protein (lane 2) or ≈50-kDa EFγ protein (lane 8). Lanes 1 and 7, vector control transfectants. Immunoprecipitation (IP) with antitag antibodies (V5 and EXP) or control normal serum (NRS) was performed from lysates, followed by immunoblot analysis with anti-V5 (lanes 3–6) or anti-EXP antibody (lanes 9–12). (F) Immunoprecipitation of Fez1 and EF1γ in synchronized 293 cells. Similar to Fig. 5 A–C, 293 cells were synchronized and analyzed after a 6-hr culture plus a 2-hr treatment with 40 μg/ml nocodazole (late S–G₂/M). Cellular proteins (1.5 mg) were subjected to immunoprecipitation with anti-EF1γ antibody (EF1γ) or normal rabbit serum (NR), followed by immunoblotting with anti-Fez1 antibody (wt:Fez, indicated by arrow).