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Interaction of ZPR1 with translation elongation factor-1alpha in proliferating cells.

Gangwani L, Mikrut M, Galcheva-Gargova Z, Davis RJ - J. Cell Biol. (1998)

Bottom Line: The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation.Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth.Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

ABSTRACT
The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1alpha (eEF-1alpha). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1alpha and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH2-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1alpha contributes to normal cellular proliferation.

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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.
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Figure 8: 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.

Mentions: The interaction of cZPR1 with eEF-1α did not appear to be essential for viability of the yeast S. cerevisiae (Table IV). However, ZPR1 and eEF-1α interact in response to extracellular growth stimuli (Fig. 4, B and C). Therefore, it was possible that this interaction may play a role in the normal process of cellular proliferation. To examine the function of the ZPR1/eEF-1α complex in cellular growth, we investigated the effect of cZPR1 mutations that disrupt eEF-1α binding on the growth of S. cerevisiae. Control experiments demonstrated that the cZPR1 mutants D1 and D2, which bind eEF-1α, exhibited no growth defect (Fig. 8 A). In contrast, the yeast strains (Table II) expressing mutant cZPR1 (D3, D5, and CT) proteins that do not bind eEF-1α (Table IV) were found to grow at least 20-fold slower than yeast expressing wild-type cZPR1 (Fig. 8, A and B).


Interaction of ZPR1 with translation elongation factor-1alpha in proliferating cells.

Gangwani L, Mikrut M, Galcheva-Gargova Z, Davis RJ - J. Cell Biol. (1998)

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.
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Figure 8: 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.
Mentions: The interaction of cZPR1 with eEF-1α did not appear to be essential for viability of the yeast S. cerevisiae (Table IV). However, ZPR1 and eEF-1α interact in response to extracellular growth stimuli (Fig. 4, B and C). Therefore, it was possible that this interaction may play a role in the normal process of cellular proliferation. To examine the function of the ZPR1/eEF-1α complex in cellular growth, we investigated the effect of cZPR1 mutations that disrupt eEF-1α binding on the growth of S. cerevisiae. Control experiments demonstrated that the cZPR1 mutants D1 and D2, which bind eEF-1α, exhibited no growth defect (Fig. 8 A). In contrast, the yeast strains (Table II) expressing mutant cZPR1 (D3, D5, and CT) proteins that do not bind eEF-1α (Table IV) were found to grow at least 20-fold slower than yeast expressing wild-type cZPR1 (Fig. 8, A and B).

Bottom Line: The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation.Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth.Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

ABSTRACT
The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1alpha (eEF-1alpha). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1alpha and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH2-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1alpha contributes to normal cellular proliferation.

Show MeSH
Related in: MedlinePlus