ZPR1 is an essential gene in the yeast S. cerevisiae. (A) The S. cerevisiae diploid WT strain (CY246) was used to create the heterozygous strain MY2 (ZPR1/zpr1::LEU2). The structures of the wild-type and disrupted cZPR1 genes are illustrated. (B) Disruption of one ZPR1 allele was confirmed by Southern blot analysis of PvuI restricted genomic DNA isolated from the diploid WT strain CY246 and the heterozygous strain MY2 (ZPR1/zpr1::LEU2). (C) The heterozygous diploid strain MY2 (ZPR1/zpr1::LEU2) was sporulated and eight randomly selected asci were dissected and examined for viability on YEPD agar media.

Coimmunoprecipitation analysis demonstrates that eEF-1α and ZPR1 interact in vivo. (A) COS-7 cells were mock-transfected (CONTROL) or transfected with a vector expressing epitope-tagged ZPR1. Flag-ZPR1 was immunoprecipitated with the M2 monoclonal antibody. eEF-1α in the immunoprecipitates (IP) was detected by protein immunoblot analysis with a monoclonal antibody to eEF-1α. (B) Coimmunoprecipitation of ZPR1 with eEF-1α from cells treated with serum or EGF. A431 epidermoid carcinoma cells were incubated in serum-free medium for 24 h. The cells were untreated or treated with 5% calf serum, 100 nM EGF, or 100 nM PMA for 15 min. Extracts were prepared from the cells and eEF-1α was isolated by immunoprecipitation. Specificity of the immunoprecipitation was examined using mock immunoprecipitates prepared using protein A–Sepharose beads without antibody to eEF-1α (CONTROL). ZPR1 in the immunoprecipitates was detected by protein immunoblot analysis using a rabbit polyclonal antibody. (C) EGF causes the rapid formation of ZPR1/eEF-1α complexes that are detected by coimmunoprecipitation analysis. A431 cells were serum starved for 24 h. The cells were incubated with 100 nM EGF for various times, extracts were prepared, and then eEF-1α was isolated by immunoprecipitation. Specificity of the immunoprecipitation was examined using mock immunoprecipitates prepared using protein A–Sepharose beads without antibody to eEF-1α (CONTROL). The presence of ZPR1 in the immunoprecipitates was examined by protein immunoblot analysis with a rabbit polyclonal antibody.

Redistribution of ZPR1 in the yeast S. cerevisiae. The haploid yeast strain MY28 (Table III) examined by fluorescence microscopy. This yeast strain expresses cZPR1–GFP in the absence of wild-type cZPR1. The cells were grown in synthetic medium with glucose (CONTROL) and starved in glucose-free medium for 12 h (STARVED). Glucose (2%) was added to the starved cells and incubated for 2 h (GLUCOSE). The GFP (green) and DAPI (blue) represents ZPR1 and DNA, respectively. Bar, 10 μm.

Mutational analysis of the effect of ZPR1 on the growth of the yeast S. cerevisiae. (A) The growth of haploid yeast strains (zpr1::LEU2) complemented with plasmid expression vectors for wild-type (WT) and mutated (D1, D2, D3, D5, and NT) cZPR1 was examined. Liquid cultures of the yeast (0.2 OD₆₀₀) were serially diluted to 5-, 10-, 50-, 100- and 1,000-fold, spotted onto YEPD plates, and then incubated at 30°C. (B) The growth of haploid yeast strains (zpr1::LEU2) complemented with plasmid expression vectors for wild-type (WT) and mutated (CT, NT, and NTΔD5) cZPR1 was examined as described above. (C) Morphology of haploid yeast strains (zpr1::LEU2) complemented with plasmid expression vectors for WT cZPR1 (strain LY1) and mutant D5 cZPR1 (strain LY6) was examined by microscopy using Nomarski optics. DNA was detected by DAPI staining and fluorescence microscopy. (D) FACS^® analysis of the haploid yeast strains LY1 and LY6 expressing wild-type and mutant (D5) cZPR1, respectively. (E) Mutational analysis of the sub-cellular localization of cZPR1 in S. cerevisiae. The location of wild-type cZPR1 (cZPR1-GFP), D5 mutant cZPR1 (D5-GFP), the NT fragment of cZPR1 (NT-GFP), and the CT fragment of cZPR1 (CT-GFP) was examined by fluorescence microscopy of yeast strains MY28, LY11, LY12, and LY14, respectively (Table II). Bars: (C) 50 μm; (E) 10 μm.

Identification of ZPR1 homologues in mammals and yeast. (A) The sequence of the S. cerevisiae, S. pombe, and mouse ZPR1 proteins deduced from the nucleotide sequence of DNA clones is presented. Residues identical to the sequence of mouse ZPR1 are indicated with a period (.). Gaps were introduced into the sequences to optimize the alignment (-). Overline, zinc fingers; asterisks, Cys residues. The sequences of mouse ZPR1, S. pombe zpr1 and S. cerevisiae ZPR1 have been deposited with GenBank/EMBL/DDBJ accession numbers U41287, AF019768 and AF019769, respectively. (B) Schematic representation of the predicted domain structure of ZPR1. The regions required for interaction with the EGF receptor are indicated (EGF-R).

ZPR1 binds to eEF-1α in vitro. (A) A431 cells were labeled with [³⁵S]methionine. Soluble extracts were prepared and incubated with immobilized ZPR1 (GST–ZPR1) proteins from three species: S. cerevisiae, S. pombe, and mouse. Bound proteins were examined by SDS-PAGE and autoradiography. Arrows, binding of the 180-kD EGF receptor to mouse ZPR1 and the prominent 50-kD protein that binds to mouse and yeast ZPR1 proteins. (B) Deletion analysis of mZPR1 sequences required for interaction with eEF-1α. Immobilized GST and GST–mZPR1 fusion proteins were incubated with cell lysates. Bound eEF-1α was detected by protein immunoblot analysis using a monoclonal antibody to eEF-1α. The binding of eEF-1α to full-length (lane 8) and truncated (lanes 1–7) mZPR1 proteins is presented. The mZPR1 amino acid residues in the truncated constructs are: 1–99 (1); 94–268 (2); 263–306 (3); 302–469 (4); 1–268 (5); 263–469 (6); 1–306 (7); and 1–469 (8). (C) Recombinant eEF-1α binds to mZPR1 in vitro. Immobilized GST and GST–eEF-1α were incubated with cell lysates. Bound mZPR1 was detected by protein immunoblot analysis using a rabbit polyclonal antibody to mZPR1. The multiple bands are characteristic of the electrophoretic mobility of mZPR1 (Galcheva-Gargova et al., 1996). (D) Binding of recombinant eEF-1α and cZPR1. Immobilized GST and GST–eEF-1α were incubated with purified CBP–cZPR1. Bound cZPR1 was detected by protein immunoblot analysis.

Redistribution of eEF-1α and ZPR1 from the cytoplasm to the nucleus in EGF-treated cells. A431 cells were incubated in serum-free medium for 24 h (starved) and then treated with 100 nM EGF or 100 nM PMA for 15 min at 37°C. The cells were fixed, permeabilized, and then examined by double-label immunofluorescence microscopy. Fluorescein (green) and cyanine (red) represent eEF-1α and ZPR1, respectively. Yellow, colocalization of eEF-1α and ZPR1 in the nucleus upon EGF treatment in merged images. Bar, 50 μm.

A small region of ZPR1 is required for interaction with eEF-1α in the yeast S. cerevisiae. (A) Schematic representation of S. cerevisiae ZPR1. NH₂-terminal (NT; residues 1–261) and COOH-terminal (CT; residues 262–486) fragments of cZPR1 are illustrated. In-frame deletions within the full-length cZPR1 protein were constructed: D1 (residues 222–241), D2 (residues 222– 261), D3 (residues 202–241), D4 (residues 202–261), and D5 (residues 202–221). The sequence of the D5 region in mouse and yeast ZPR1 proteins is illustrated. (B–D) Recombinant cZPR1 proteins were expressed as a CBP fusion protein in bacteria. The CBP fusion proteins were purified, immobilized, and then used for binding assays using cell lysates. Bound eEF-1α was detected by protein immunoblot analysis. The binding of eEF-1α to NT and CT fragments of cZPR1 was examined (B). The effect of in-frame deletions (D1, D2, D3, and D4) of cZPR1 was investigated (C). The effect of the D5 in-frame deletion mutation of cZPR1 was also examined (D).