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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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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.
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Figure 5: 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.

Mentions: We examined the subcellular localization of ZPR1 and eEF-1α in mammalian cells by double-label immunofluorescence microscopy. Consistent with previous studies (Galcheva-Gargova et al., 1996) we observed that ZPR1 (red) redistributed from the cytoplasm to the nucleus upon EGF stimulation of serum-starved cells (Fig. 5). eEF-1α is an abundant protein that is present mainly in the cytoplasm of cells (Edmonds et al., 1996). Fig. 5 shows the effect of EGF treatment on the subcellular distribution of eEF-1α (green). In serum-starved cells, eEF-1α was present in the cytoplasm. Upon treatment with EGF, a fraction of the eEF-1α molecules was observed to redistribute from the cytoplasm to the nucleus. The distribution of eEF-1α and ZPR1 in the nucleus after treatment of cells with EGF was similar, as represented by yellow fluorescence in the merged images of ZPR1 and eEF-1α (Fig. 5). Similar results were obtained upon treatment of serum-starved cells with 10% calf serum (data not shown). Quantitative analysis of the fluorescence images demonstrated that ZPR1 was ∼98% cytoplasmic in serum-starved cells and was ∼96% nuclear in EGF-treated cells. Similar analysis demonstrated that eEF-1α was not detected in the nucleus of serum-starved cells and that ∼5% of the total eEF-1α was located in the nucleus after stimulation with EGF. These data indicate that both ZPR1 and eEF-1α are located in the nucleus of mitogen-treated cells. The punctate distribution of ZPR1 in the nucleus was previously demonstrated to correspond to an accumulation within nucleoli (Galcheva-Gargova et al., 1996). This nucleolar accumulation is characterized by colocalization of ZPR1 with fibrillarin and RNA polymerase I, but not with splicing factor SC35 or p80 coilin (Galcheva-Gargova et al., 1998).


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

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

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132977&req=5

Figure 5: 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.
Mentions: We examined the subcellular localization of ZPR1 and eEF-1α in mammalian cells by double-label immunofluorescence microscopy. Consistent with previous studies (Galcheva-Gargova et al., 1996) we observed that ZPR1 (red) redistributed from the cytoplasm to the nucleus upon EGF stimulation of serum-starved cells (Fig. 5). eEF-1α is an abundant protein that is present mainly in the cytoplasm of cells (Edmonds et al., 1996). Fig. 5 shows the effect of EGF treatment on the subcellular distribution of eEF-1α (green). In serum-starved cells, eEF-1α was present in the cytoplasm. Upon treatment with EGF, a fraction of the eEF-1α molecules was observed to redistribute from the cytoplasm to the nucleus. The distribution of eEF-1α and ZPR1 in the nucleus after treatment of cells with EGF was similar, as represented by yellow fluorescence in the merged images of ZPR1 and eEF-1α (Fig. 5). Similar results were obtained upon treatment of serum-starved cells with 10% calf serum (data not shown). Quantitative analysis of the fluorescence images demonstrated that ZPR1 was ∼98% cytoplasmic in serum-starved cells and was ∼96% nuclear in EGF-treated cells. Similar analysis demonstrated that eEF-1α was not detected in the nucleus of serum-starved cells and that ∼5% of the total eEF-1α was located in the nucleus after stimulation with EGF. These data indicate that both ZPR1 and eEF-1α are located in the nucleus of mitogen-treated cells. The punctate distribution of ZPR1 in the nucleus was previously demonstrated to correspond to an accumulation within nucleoli (Galcheva-Gargova et al., 1996). This nucleolar accumulation is characterized by colocalization of ZPR1 with fibrillarin and RNA polymerase I, but not with splicing factor SC35 or p80 coilin (Galcheva-Gargova et al., 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.

Show MeSH
Related in: MedlinePlus