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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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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.
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Figure 2: 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.

Mentions: We examined whether the gene encoding cZPR1 was essential for cell viability in S. cerevisiae. A DNA fragment containing the cZPR1 gene disrupted by the LEU2 gene (Fig. 2 A) was transformed into the diploid yeast strain CY246. Colonies were selected on plates lacking leucine and the disruption of one genomic copy of cZPR1 was confirmed by Southern blot analysis (Fig. 2 B). The diploid yeast strain MY2, which is heterozygous for wild-type cZPR1 (ZPR1/zpr1::LEU2), was sporulated and the tetrads were dissected. In all tetrads analyzed, the viable progeny segregated 2:2, and none of the surviving spores were Leu+ (disrupted for cZPR1). These data indicate that the cZPR1 gene is essential for viability (Fig. 2 C). To establish that the loss of viability was due to the disruption of the cZPR1 gene, we complemented haploid yeast with the disrupted cZPR1 gene (zpr1::LEU2) using a plasmid expressing wild-type cZPR1. These data demonstrated that the cZPR1 gene was essential for viability in S. cerevisiae.


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

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

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.
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Related In: Results  -  Collection

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Figure 2: 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.
Mentions: We examined whether the gene encoding cZPR1 was essential for cell viability in S. cerevisiae. A DNA fragment containing the cZPR1 gene disrupted by the LEU2 gene (Fig. 2 A) was transformed into the diploid yeast strain CY246. Colonies were selected on plates lacking leucine and the disruption of one genomic copy of cZPR1 was confirmed by Southern blot analysis (Fig. 2 B). The diploid yeast strain MY2, which is heterozygous for wild-type cZPR1 (ZPR1/zpr1::LEU2), was sporulated and the tetrads were dissected. In all tetrads analyzed, the viable progeny segregated 2:2, and none of the surviving spores were Leu+ (disrupted for cZPR1). These data indicate that the cZPR1 gene is essential for viability (Fig. 2 C). To establish that the loss of viability was due to the disruption of the cZPR1 gene, we complemented haploid yeast with the disrupted cZPR1 gene (zpr1::LEU2) using a plasmid expressing wild-type cZPR1. These data demonstrated that the cZPR1 gene was essential for viability in S. cerevisiae.

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