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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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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).
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Figure 7: 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).

Mentions: To examine the interaction of ZPR1 with eEF-1α, we performed deletion analysis of cZPR1. Studies using NH2-terminal (NT) and COOH-terminal (CT) fragments of cZPR1 demonstrated that the eEF-1α–binding site was located in the NH2-terminal region of cZPR1 (residues 1–261) (Fig. 7, A and B). We constructed a series of COOH-terminal truncated mutants of the NT fragment of cZPR1. Binding assays indicated that cZPR1 residues 202–261 were required for the interaction of eEF-1α with cZPR1 (data not shown). Subsequently, a series of in-frame deletion mutants of full-length cZPR1 (D1-D4) was prepared (Fig. 7 A). These internal deletion mutants were expressed as CBP fusions in bacteria and purified. The binding of eEF-1α to these cZPR1 proteins was detected by immunoblot analysis. The in-frame deletions D1 (residues 222– 241) and D2 (residues 222–261) had no effect on eEF-1α binding, whereas deletions D3 (residues 202–241) and D4 (residues 202–261) eliminated the binding of eEF-1α to cZPR1 (Fig. 7 C). These data suggest that a sequence required for eEF-1α binding is present in the D3 region (residues 202–241) of cZPR1 (Fig. 7 A). To further delineate the cZPR1 sequence required for the binding of eEF-1α, the smaller in-frame deletion mutant D5 (residues 202– 221) was prepared and tested for eEF-1α binding. Immunoblot analysis demonstrated that the cZPR1 deletion mutant D5 did not bind eEF-1α (Fig. 7 D). Therefore, the 20-amino acid region D5 (residues 202–221) was required to be present in full-length and truncated ZPR1 for eEF-1α binding. This region is located within the A-domain of the cZPR1 protein and is conserved in mouse, S. cerevisiae and S. pombe (Fig. 7 A).


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

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

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).
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Figure 7: 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).
Mentions: To examine the interaction of ZPR1 with eEF-1α, we performed deletion analysis of cZPR1. Studies using NH2-terminal (NT) and COOH-terminal (CT) fragments of cZPR1 demonstrated that the eEF-1α–binding site was located in the NH2-terminal region of cZPR1 (residues 1–261) (Fig. 7, A and B). We constructed a series of COOH-terminal truncated mutants of the NT fragment of cZPR1. Binding assays indicated that cZPR1 residues 202–261 were required for the interaction of eEF-1α with cZPR1 (data not shown). Subsequently, a series of in-frame deletion mutants of full-length cZPR1 (D1-D4) was prepared (Fig. 7 A). These internal deletion mutants were expressed as CBP fusions in bacteria and purified. The binding of eEF-1α to these cZPR1 proteins was detected by immunoblot analysis. The in-frame deletions D1 (residues 222– 241) and D2 (residues 222–261) had no effect on eEF-1α binding, whereas deletions D3 (residues 202–241) and D4 (residues 202–261) eliminated the binding of eEF-1α to cZPR1 (Fig. 7 C). These data suggest that a sequence required for eEF-1α binding is present in the D3 region (residues 202–241) of cZPR1 (Fig. 7 A). To further delineate the cZPR1 sequence required for the binding of eEF-1α, the smaller in-frame deletion mutant D5 (residues 202– 221) was prepared and tested for eEF-1α binding. Immunoblot analysis demonstrated that the cZPR1 deletion mutant D5 did not bind eEF-1α (Fig. 7 D). Therefore, the 20-amino acid region D5 (residues 202–221) was required to be present in full-length and truncated ZPR1 for eEF-1α binding. This region is located within the A-domain of the cZPR1 protein and is conserved in mouse, S. cerevisiae and S. pombe (Fig. 7 A).

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