The Zinc Finger Protein ZPR1 Is Conserved in Mammals and Yeast
Homologues of the mammalian zinc finger protein ZPR1 were identified and cloned from the yeast S. cerevisiae and Schizosaccharomyces pombe. The sequence of the mouse, S. cerevisiae and S. pombe ZPR1 proteins was deduced from the nucleotide sequence of DNA clones (Fig. A). Comparison of these ZPR1 proteins indicates a conserved structure including two similar zinc fingers (ZnF1 and ZnF2) and similar regions after each zinc finger (Fig. B, A and B). The mouse ZPR1 (mZPR1) protein is 46% identical and 72% similar to S. cerevisiae ZPR1 (cZPR1) and 43% identical and 70% similar to S. pombe ZPR1 (pZPR1). The two yeast proteins are 55% identical and 78% similar. The high level of homology between yeast and mammalian ZPR1 suggests that the overall structure of the protein is conserved between lower and higher eukaryotes.
Figure 1 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 (more ...)
ZPR1 Is an Essential Gene in S. cerevisiae
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. 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. 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. 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.
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 (more ...)
Identification of eEF-1α as a ZPR1-binding Protein
To study the interaction of ZPR1 with other proteins, we performed in vitro binding assays using [35S]methionine human A431 epidermoid carcinoma cell extracts and immobilized GST–ZPR1 fusion proteins. As expected, the mouse ZPR1 protein bound to the 180-kD EGF receptor (Fig. A). In addition, several other [35S]methionine proteins were observed to bind ZPR1. In particular, a 50-kD protein was prominently detected in binding assays using mouse, S. cerevisiae and S. pombe ZPR1 proteins (Fig. A). Similar results were obtained in binding assays using [35S]methionine extracts prepared from COS-7, CHO, and S. cerevisiae (strain L40) cells (data not shown). These data indicate that the yeast and mammalian ZPR1 proteins may have similar biochemical properties, including interaction with a 50-kD protein.
Figure 3 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 (more ...)
Deletion analysis of mouse ZPR1 using [35S]methionine cell extracts demonstrated that an NH2-terminal mZPR1 fragment (ZnF1-A; residues 1–268) was sufficient for binding the 50-kD protein (data not shown). This fragment of mZPR1 was used to isolate the 50-kD protein from human (A431) and S. cerevisiae (L40) cell extracts by affinity chromatography. The bound proteins were resolved by SDS-PAGE and electrotransferred onto a PVDF membrane. The 50-kD protein was digested with l-tosylamido-2-phenyl ethyl chloromethyl ketone-treated trypsin and examined by mass spectroscopy using the MALDI-TOF technique. A comparison of the masses of the polypeptides obtained with the MS-Fit data base led to the identification of the 50-kD protein as eEF-1α (Table ).
Identification of the 50-kD ZPR1-binding Protein by Mass Spectroscopy
To confirm the identification of the 50-kD protein as eEF-1α, we examined the proteins that bound to mZPR1 by Western blot analysis using an antibody to eEF-1α. Immobilized recombinant mZPR1 was found to bind eEF-1α (Fig. B, lane 8). Similarly, immobilized recombinant eEF-1α bound to mZPR1 (Fig. C). Deletion analysis demonstrated that the NH2-terminal region of mZPR1 was sufficient for binding eEF-1α (Fig. B). Deletion analysis of eEF-1α demonstrated that the NH2-terminal region (residues 1–300) was required for binding ZPR1 (data not shown). To test whether ZPR1 and eEF-1α bind directly, we performed in vitro assays using the two purified recombinant proteins. This analysis demonstrated that eEF-1α directly binds to cZPR1 (Fig. D).
Interaction of eEF-1α and ZPR1 In Vivo
To test whether eEF-1α and ZPR1 may interact in vivo, we performed coimmunoprecipitation analysis. Epitope-tagged mZPR1 was immunoprecipitated from serum-treated COS-7 cells and the presence of eEF-1α in the immunoprecipitates was examined by Western blot analysis. Fig. A shows that eEF-1α was coimmunoprecipitated with ZPR1. Control experiments using cells that did not express epitope-tagged mZPR1 demonstrated no coimmunoprecipitation of eEF-1α. These data indicate that ZPR1 and eEF-1α may interact in vivo.
Figure 4 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 (more ...)
ZPR1 has been shown to bind the EGF receptor in quiescent cells and to be released from the receptor upon treatment with EGF (Galcheva-Gargova et al. 1996
). EGF may therefore regulate the interaction of ZPR1 with eEF-1α. To examine the effect of EGF, we starved A431 cells in serum-free medium for 24 h and then treated the cells with 100 nM EGF. eEF-1α was immunoprecipitated and the presence of ZPR1 was detected by Western blot analysis. Coimmunoprecipitation of ZPR1 with eEF-1α was detected in the EGF-treated cells, but not in the serum-starved cells (Fig. B
). The amount of coimmunoprecipitated ZPR1 was sustained during longer periods of treatment with EGF (Fig. C
). These data indicate that EGF induces an interaction between ZPR1 and eEF-1α. To test whether this interaction between ZPR1 and eEF-1α can be induced by other stimuli, we studied the effect of serum. Treatment of serum-starved cells with fetal calf serum (10%) resulted in the coimmunoprecipitation of ZPR1 with eEF-1α (Fig. , B
). Based on these observations, we propose that growth signals in mitogen-activated cells may induce the formation of eEF-1α/ZPR1 complexes in vivo. Specificity in the formation of the eEF-1α/ ZPR1 complex was established by the observation that treatment of the cells with the tumor promoter phorbol 12-myristate 13-acetate (PMA) did not cause coimmunoprecipitation of ZPR1 with eEF-1α (Fig. B
Redistribution of ZPR1 and eEF-1α to the Nucleus of Mitogen-activated Mammalian Cells
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. ). eEF-1α is an abundant protein that is present mainly in the cytoplasm of cells (Edmonds et al., 1996
). Fig. 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. ). 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
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 (more ...)
We studied the effect of PMA on the subcellular distribution of eEF-1α and ZPR1 to confirm the specificity of the interaction between eEF-1α and ZPR1 in the response to extracellular stimulation. PMA is known to induce the phosphorylation of eEF-1α by activating protein kinase C (Venema et al., 1991
). However, PMA did not cause punctate nuclear accumulation of eEF-1α or ZPR1 (Fig. ). These data are consistent with the results of coimmunoprecipitation analysis where treatment with PMA did not induce the formation of ZPR1/eEF-1α complexes (Fig. B
). We conclude that ZPR1 responds to selective extracellular stimuli.
Nuclear Redistribution of ZPR1 in Yeast
To study the localization of cZPR1 in the yeast S. cerevisiae, a recombinant cZPR1–GFP fusion gene was constructed. The cZPR1–GFP gene complemented the loss of viability of yeast with the disrupted zpr1::LEU2 gene. These haploid yeast expressing the cZPR1–GFP fusion protein were examined by fluorescence microscopy. Control experiments demonstrated that GFP was sequestered in the vacuole of yeast expressing GFP alone (data not shown). Fig. shows that the cZPR1–GFP fusion protein (green) was present mainly in the nucleus of S. cerevisiae (CONTROL). Starvation of the yeast in glucose-free medium caused the redistribution of cZPR1 from the nucleus to the cytoplasm (Fig. ). Readdition of glucose to the starved cells induced the translocation of cZPR1 from the cytoplasm to the nucleus. The localization and redistribution of ZPR1 in response to nutrients in S. cerevisiae appears to be similar to that observed in starved and mitogen-treated mammalian cells. Therefore, it is possible that cZPR1 may function as a signaling molecule in S. cerevisiae that responds to proliferation signals.
Figure 6 Redistribution of ZPR1 in the yeast S. cerevisiae. The haploid yeast strain MY28 (Table ) examined by fluorescence microscopy. This yeast strain expresses cZPR1–GFP in the absence of wild-type cZPR1. The cells were grown in synthetic (more ...)
Identification of Mutant ZPR1 Molecules That Fail to Interact with eEF-1α
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. , 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. 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. C). These data suggest that a sequence required for eEF-1α binding is present in the D3 region (residues 202–241) of cZPR1 (Fig. 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. 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. A).
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 (more ...)
Mutational Analysis of ZPR1 Function
We examined the effect of cZPR1 mutations that disrupt eEF-1α binding on the viability of S. cerevisiae. The in-frame deletion mutants D1, D2, D3, D4, and D5 complemented the loss of viability caused by the disruption of the cZPR1 gene in S. cerevisiae (Table ). These data indicate that deletion of sequences required for eEF-1α binding did not affect the essential role of ZPR1 required for viability. Therefore, the binding of cZPR1 to eEF-1α was not required for viability of S. cerevisiae.
Mutational Analysis of the Effect of ZPR1 on Yeast Viability and the Interaction of ZPR1 with eEF-1α
We performed deletion analysis to identify the region of cZPR1 that was required for yeast viability. Expression of an NT fragment of cZPR1 (residues 1–261), which includes the first zinc finger and the region required for interaction with eEF-1α, did not confer viability (Table ). In contrast, expression of a CT fragment of cZPR1 (residues 262–486), which includes the second zinc finger and does not interact with eEF-1α, was able to confer viability (Table ). These data demonstrate that the functions of cZPR1 to bind eEF-1α and to confer viability are encoded within separate regions of the cZPR1 gene. eEF-1α binding requires the NT fragment of cZPR1 and viability requires the CT fragment of cZPR1.
To further examine the sequences present in the CT fragment of cZPR1 that are required for viability, we examined the effect of mutations in the cZPR1 zinc fingers. The zinc fingers were disrupted by replacing two of the Cys residues with Ala. Mutation of zinc finger 1 (located in the NT fragment of cZPR1) complemented the loss of viability caused by disruption of the ZPR1 gene. In contrast, mutation of the second zinc finger (present in the CT region of cZPR1) did not confer viability in this assay (Table ). These data indicate that the sequences located in CT that are required for viability include the second zinc finger.
Interaction of ZPR1 with eEF-1α Is Required for Normal Growth
The interaction of cZPR1 with eEF-1α did not appear to be essential for viability of the yeast S. cerevisiae (Table ). However, ZPR1 and eEF-1α interact in response to extracellular growth stimuli (Fig. , 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. A). In contrast, the yeast strains (Table ) expressing mutant cZPR1 (D3, D5, and CT) proteins that do not bind eEF-1α (Table ) were found to grow at least 20-fold slower than yeast expressing wild-type cZPR1 (Fig. , A and B).
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. (more ...)
The morphology of the yeast strains expressing wild-type and mutant cZPR1 proteins was examined by light microscopy. Fig. C shows images of yeast expressing wild-type cZPR1 or mutant D5 cZPR1 that does not bind eEF-1α. Cells with mutant cZPR1 (D5) are larger and grow in chain-like clusters. The failure of the cells to separate and their larger size suggest that these slow-growing yeast may accumulate in the G2/M phase of the cell cycle. These cells were therefore examined for DNA content by flow cytometry. Fig. C shows that the wild-type cells are distributed between the G1 and G2 phases of the cell cycle, corresponding to 1N and 2N DNA content, respectively (Fig. C). In contrast, the majority of the mutant yeast cells contained 2N DNA (Fig. D), consistent with an accumulation in the G2/M phase of the cell cycle (Fig. C). These data suggest that the interaction of cZPR1 with eEF-1α may be required for normal cell cycle progression.
To examine whether the absence of binding to eEF-1α is relevant to the defective growth of yeast strains that express mutant cZPR1 molecules (D3, D5, and CT), we performed an interallelic complementation assay. The NT fragment of cZPR1 binds eEF-1α, but is not sufficient for viability. However, the expression of the NT fragment of cZPR1 in the D3, D5, and CT mutant ZPR1 yeast strains restored normal proliferation (Fig. , A and B). To confirm that the restoration of normal growth is due to the interaction of ZPR1 with eEF-1α, a yeast strain expressing the CT fragment was transformed with a plasmid expressing the NT (ΔD5) fragment of cZPR1 which does not bind eEF-1α. Expression of NT (ΔD5) did not restore the growth of the yeast complemented with the CT fragment of cZPR1 (Fig. B). These data are consistent with the hypothesis that the interaction of cZPR1 with eEF-1α was required for the normal growth of yeast. The specificity of the D5 mutation to cause reduced growth was tested by comparison with the effect of mutation of the first zinc finger of cZPR1 (replacement of two of the Cys residues with Ala). Mutation of the first zinc finger caused slow growth that was not associated with accumulation at the G2/M phase of the cell cycle (data not shown). Thus, the G2/M accumulation caused by the D5 mutation does not represent a nonspecific consequence of defects in cZPR1 function.
To further examine the effect of the NT fragment of cZPR1 to restore the normal growth of yeast that express the D5 mutant cZPR1, we studied the effect of cZPR1 mutations on the subcellular localization of cZPR1 in S. cerevisiae. The GFP-labeled NT and CT fragments of cZPR1 (NT–GFP and CT–GFP), like wild-type cZPR1-GFP, were found to accumulate within the nucleus (Fig. E). The distribution of the D5 mutant cZPR1-GFP (D5–GFP) was similar to wild-type cZPR1–GFP (Fig. E). Therefore, deletion of the D5 region did not affect the nuclear distribution of cZPR1 in growing cells. These data demonstrate that cZPR1 can accumulate in the nucleus independently of eEF-1α.