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Genetics, Vol. 171, 1571-1581, December 2005, Copyright © 2005
doi:10.1534/genetics.105.048082
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Department of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University, UNESP, Araraquara, São Paulo 14801-902, Brazil
1 Corresponding author: School of Pharmaceutical Sciences, São Paulo State University, UNESP, Rodovia Araraquara-Jaú, Km 01, Araraquara, SP 14801-902, Brazil.
E-mail: valentsr{at}fcfar.unesp.br
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ABSTRACT |
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The involvement of eIF5A with translation initiation was proposed due to its purification from ribosomes of reticulocyte lysates and to its stimulatory effect in the methionyl-puromycin assay used to implicate factors in the first peptide bond formation (BENNE and HERSHEY 1978). However, depletion of eIF5A in yeast caused only a slight decrease in the protein synthesis rate, arguing against a role as a general translation initiation factor. Therefore, it was hypothesized that eIF5A may function in the translation of a specific subset of mRNAs. Since depletion of eIF5A in yeast also causes an increase of G1-arrested cells, judged by cell morphology, it was proposed that eIF5A may be important for translating mRNAs encoding proteins required for cell cycle progression (KANG and HERSHEY 1994). This connection between eIF5A and cell cycle progression is further supported by the observation that blocking any step of hypusination, its essential post-translational modification, in mammalian cells inhibits cell proliferation (PARK et al. 1997), placing eIF5A among the potential targets for cancer therapy (CARAGLIA et al. 2001).
eIF5A has also been implicated in nucleocytoplasmic export of Rev-dependent HIV-1 transcripts and mRNA decay (RUHL et al. 1993; BEVEC et al. 1996; BEVEC and HAUBER 1997; ZUK and JACOBSON 1998). However, subsequent studies have not confirmed the involvement of eIF5A with Rev-dependent nuclear export in either mammalian or yeast systems (SHI et al. 1996, 1997; HENDERSON and PERCIPALLE 1997; LIPOWSKY et al. 2000; LI-EN JAO and CHEN 2002; VALENTINI et al. 2002). Moreover, the effect of eIF5A on mRNA decay seems to be secondary, as arrest of cell growth of eIF5A temperature-sensitive mutants does not directly correlate with mRNA accumulation (VALENTINI et al. 2002).
Thus, although eIF5A has been associated with different cellular events, the role played by this essential factor remains unclear. In an attempt to identify cellular partners for eIF5A and understand its critical cellular function, a temperature-sensitive mutant of TIF51A (tif51A-1), one of the genes encoding eIF5A in Saccharomyces cerevisiae, was used in a high-copy suppressor screen. PKC1, encoding for the only yeast protein kinase C, was one of the suppressors isolated together with three members of the cell integrity pathway, WSC1, WSC2, and WSC3 (VALENTINI et al. 2002).
Pkc1 in S. cerevisiae controls a variety of cellular processes such as cell cycle progression, mating, nutrient sensing, and the structural organization of the cytoskeleton (HEINISCH et al. 1999). Pkc1 is activated by a small GTPase of the Rho family, Rho1, which receives upstream signals from the Slg1 (Wsc1) and Mid2 transmembrane sensors (PHILIP and LEVIN 2001). The downstream PKC1-mitogen-activated protein kinase (MAPK) cascade, consisting of Bck1, Mkk1/Mkk2, and Mpk1 kinases, phosphorylates transcription factors that regulate cell wall remodeling and cytoskeleton organization in polarized cell growth (HEINISCH et al. 1999). Although less well defined, several studies report evidence for other biochemical pathways branching out from Pkc1 (KETELA et al. 1999; ANDREWS and STARK 2000; LI et al. 2000; NANDURI and TARTAKOFF 2001; CHAI et al. 2002; VALDIVIA and SCHEKMAN 2003; VILELLA et al. 2005).
In this work, we further analyzed the functional interaction between eIF5A and the Pkc1-cell integrity pathway. Our results demonstrate that PKC1 suppression of tif51A-1 is MAP kinase independent. Furthermore, we present data that suggest the existence of a novel pathway independent of the MAP kinases. This pathway links Pkc1 to the Cdc42 effector, Gic1, in a Zds1/Zds2-dependent manner. The results herein favor a more direct involvement of Pkc1 in actin polarization, which is necessary for bud formation during G1/S transition. Finally, we discuss a possible role for eIF5A in this process.
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MATERIALS AND METHODS |
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Phalloidin staining:
Staining of actin filaments was carried out essentially as described (AMBERG 1998). Cells from exponential phase cultures were fixed at room temperature for 1 hr with 3.7% formaldehyde in the culture medium plus 1 hr incubation in PBS with 3.7% formaldehyde. Cells were subsequently washed twice with PBS and resuspended in 500 µl of PBS. Staining was performed by adding 10 µl of rhodamine-phalloidin (Molecular Probes, Eugene, OR) to 100 µl of cell suspension. Cells were incubated in the dark for 1 hr, washed five times with 1 ml of PBS, and finally suspended in 100 µl of mount solution [90% glycerol, 0.1x PBS, 92.5 mM p-phenylenediamine (Sigma, St. Louis), pH adjusted to 0.8 with 0.5 M sodium carbonate, pH 9.0]. Stained cells were stored at –20° until microscopic analysis. Rhodamine-phalloidin-stained cells (2.5 µl) were visualized by fluorescence microscopy using a rhodamine filter and a Nikon TE300 inverted microscope. Images were captured with a MicroMax 5-MHz CCD (Princeton Instruments, Princeton, NJ) and the software Image-Pro Plus (Media Cybernetics).
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RESULTS |
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To determine whether the suppression of tif51A-1 promoted by high-copy PKC1 occurs via the MAP kinases, we examined whether high-copy MKK1, MPK1, or an activated allele of BCK1, BCK1-20, could suppress the temperature-sensitive phenotype of tif51A-1. Unlike PKC1, none of the MAP kinase genes was able to suppress the tif51A-1 growth defect at the nonpermissive temperature of 37° (Figure 1A). However, as a control, the activated allele BCK1-20 successfully overcame the defect of a PKC1 temperature-sensitive mutant, stt1 (YOSHIDA et al. 1994; Figure 1B). This result suggests that, although BCK1-20 can suppress the temperature-sensitive phenotype of the pkc1 mutant (stt1), activation of the cell integrity MAP kinase pathway is not sufficient to overcome the tif51A-1 growth phenotype. In contrast to the results with genes that function downstream of Pkc1, high-copy plasmids containing genes for known and putative upstream activators of Pkc1 (including WSC1-3, MID2, MTL1, ROM2, and RHO2) do suppress the tif51A-1 mutant phenotype (HOHMANN 2002; Table 3). Taken together, these results strongly suggest that Pkc1 does not act through its downstream MAP kinase cascade to promote suppression of the tif51A-1 temperature-sensitive phenotype.
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, or tif51A-1 and tif51A-2 at higher temperatures (data not shown), demonstrating that these suppressors can only partially correct the defects of the mutants analyzed. Gic1 is an effector of Cdc42 that is important for bud emergence and contains a Cdc42/Rac-interactive-binding (CRIB) domain, which mediates interaction with GTP-bound Cdc42 (BROWN et al. 1997). Gic1, together with its homolog Gic2, seems to exert its function during the G1/S cell cycle transition by linking the major polarization organizer Cdc42 and the formin Bni1, which is responsible for induction of actin polymerization (PRUYNE et al. 2004). Zds1 has also been implicated in cell cycle progression but with a less well-defined function (MA et al. 1996). ZDS1 and ZDS2, its functionally redundant homolog, have been isolated in a series of high-copy suppressor screens (SCHWER and SHUMAN 1996; TSUCHIYA et al. 1996; BOURBONNAIS et al. 2001; SEKIYA-KAWASAKI et al. 2002). Interestingly, high levels of Zds1 can decrease Cdc42 activity (BI and PRINGLE 1996) and high-throughput two-hybrid data have shown physical interactions among Pkc1, Zds2, Zds1, and Gic1 (DREES et al. 2001).
To further investigate the relationship among Pkc1, Zds1, and Gic1, we tested whether Zds function is important for the suppression mediated by high-copy PKC1 or GIC1. To address this question, a tif51A-1 strain lacking both ZDS1 and ZDS2 was generated (SVL411), and the ability of PKC1 or GIC1 to suppress the temperature-sensitive growth phenotype of this mutant was examined (Figure 3). To avoid possible influences of the genetic background on PKC1 suppression, this assay was performed in parallel with the strain SVL412, obtained from the same tetrad as SVL411, but containing only the tif51A-1 mutation. Interestingly, PKC1 could not suppress tif51A-1 in the absence of ZDS1 and ZDS2, while GIC1 could (Figure 3, A and B, compare rows indicated by arrows). Also, the tif51A-1 zds1 zds2 strain demonstrates enhanced temperature sensitivity, lowering from 37° to 35° the restrictive temperatures of the triple mutant (Figure 3, A and B, right). This synthetic sickness between these genes strengthens their functional connection. These data, together with the previous physical interactions described (DREES et al. 2001), strongly support a model in which Pkc1 acts through a downstream pathway different from the MAP kinase cascade. Furthermore, these data suggest that both Zds1 and Gic1 participate in this signaling pathway. Thus, as all these factors act in the same pathway to promote eIF5A mutant suppression, it is possible that eIF5A plays a role in cell polarity.
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To determine whether the interaction between Gic1 and Cdc42 is required for GIC1-mediated suppression of tif51A-1, it was tested if GIC1CRIB–, an allele known to disrupt protein-protein interactions with Cdc42 (BROWN et al. 1997), can suppress the tif51A-1 mutant. As shown in Figure 4A, GIC1 lacking the CRIB domain could not suppress tif51A-1, suggesting that although CDC42 does not suppress by itself, a Gic1-Cdc42 physical interaction may be necessary for high-copy GIC1 suppression of the tif51A-1 temperature-sensitive phenotype.
In addition to a Gic1 function in actin polarization, a high-copy suppressor screen with let1, a mitotic exit network component mutant, has recently implicated Gic1 in this process. This new role for Gic1 is separable from that in the G1/S transition, since the function in mitotic exit requires a pool of Gic1 not associated with the cell cortex, as a prenylated form of Gic1 (Gic1-pr) cannot work in the mitotic exit (HÖFKEN and SCHIEBEL 2004). To determine which Gic1 function is necessary to suppress the tif51A-1 mutant phenotype, we tested the cortex-restricted Gic1 for suppression of tif51A-1. As shown in Figure 4A, this form of Gic1 promotes growth of the tif51A-1 mutant at the nonpermissive temperature. This result supports the model that the G1/S transition function of Gic1 is responsible for the tif51A-1 mutant suppression.
The last set of genes functionally linked to GIC1 includes BEM2, BEM1, BNI1, and BUD1. These genes are involved with polarized cell growth and show genetic defects with gic1 gic2 (CHEN et al. 1997; JAQUENOUD and PETER 2000; KAWASAKI et al. 2003). We tested whether any of these genes could suppress the phenotype of the tif51A-1 mutant. This analysis revealed that both BEM1 and BNI1 are high-copy suppressors of tif51A-1 (Figure 4B; Table 3). These results show that other factors important for G1/S transition are also able to suppress the tif51A-1 mutant.
Finally, we tested whether the following ZDS1-correlated genes could suppress tif51A-1: ZDS2, its homolog; RGA1 and BEM3, encoding negative regulators of Cdc42; and SSD1, a gene of unknown function that suppresses defects in the cell integrity pathway and has its null mutant suppressed by ZDS1 (TSUCHIYA et al. 1996; KAEBERLEIN and GUARENTE 2002). Among these genes, only ZDS2 and SSD1 suppress the tif51A-1 phenotype (Figure 4B; Table 3). These results show that ZDS2 shares the ZDS1 function necessary for tif51A-1 mutant suppression. The fact that presence of SSD1 can greatly compensate for loss of eIF5A function is considered later (see DISCUSSION). The observation that neither RGA1 nor BEM3 was identified as suppressors suggests that the Zds1 function important for suppression is, most probably, not related to its role in negative regulation of Cdc42 (BI and PRINGLE 1996).
Taken together, the results point to a G1/S defect in the tif51A-1 mutant as most of the suppressors described above act in cell polarity during cell cycle progression. An important event in G1/S transition in S. cerevisiae is the establishment of an axis of polarity. Immediately after the positioning of the Cdc42-related factors at the bud site, the assembly of a polarized actin cytoskeleton is crucial for progression of the cell cycle (PRUYNE et al. 2004). Therefore, we tested whether two mutants of TIF51A, tif51A-1 and tif51A-3, exibit any actin polarization defect. Interestingly, both mutants showed marked defects in actin cytoskeleton organization in budding cells at the nonpermissive temperature. In contrast, actin cables and patches appeared normal during growth at the permissive temperature (Figure 5A).
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GIC1 overexpression can cause cell hyperpolarization:
While analyzing the effect of overexpression of GIC1 on the actin cytoskeleton defect of the tif51A-1 mutant, it was noted that 
10–20% of cells exhibited elongated buds with a hyperpolarized actin cytoskeleton (Figure 5B, GIC1, bottom). To investigate if this hyperpolarization was correlated with the tif51A-1 mutant suppression by GIC1, the morphology of cells growing on a plate at the restrictive temperature was inspected. We observed that both elongated and normal morphologies were present, but elongated ones occurred at a much lower number, apparently at the same frequency as that seen during actin cytoskeleton observation (data not shown). Furthermore, to check if cells acquiring elongated morphology are able to successfully progress through the cell cycle and thus are not sick or dead, time-lapse microscopy was performed. This analysis revealed no apparent defect in growth and, moreover, the generation of new buds was also detected (Figure 6). These data imply that actin hyperpolarization is not necessary for GIC1 suppression and that the resulting elongated cells can properly progress through the cell cycle.
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DISCUSSION |
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Moreover, consistent with the suppression data, although eIF5A mutants show enhanced growth on media containing 1 M sorbitol (VALENTINI et al. 2002), they do not demonstrate other phenotypes associated with defects in cell integrity of MAP kinase mutants such as cell lysis at the restrictive temperature and sensitivity to caffeine, staurosporine (a specific inhibitor of PKC isozymes), and calcofluor white (HEINISCH et al. 1999) (data not shown). Thus, considering the fact that PKC1 suppression is MAP kinase independent, we searched for new suppressors of the eIF5A mutant tif51A-1 that could unveil the effectors downstream of Pkc1 that are rescuing the growth impairment of this eIF5A mutant.
A novel pathway links Pkc1 to the Cdc42 effector Gic1:
In addition to the well-known tif51A-1 mutant suppressor PKC1, we show here that ZDS1 and GIC1 are also suppressors of tif51A-1. As the proteins encoded by these three genes participate in a network of physical interactions (DREES et al. 2001), we tested the hypothesis that Pkc1-Zds1-Gic1 constitutes a new signaling pathway, illustrated in the model in Figure 8. In agreement with this model, PKC1 suppression is abolished in tif51A-1 cells lacking the redundant genes ZDS1 and ZDS2. In contrast, high-copy GIC1 still suppresses the tif51A-1 zds1 zds2 triple mutant. Considering that Pkc1 acts in response to Rho1 (HEINISCH et al. 1999), this is the first study demonstrating functional data linking Rho1- and Cdc42-regulated pathways through Zds1 and Zds2.
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zds2 cells at any temperature, and it does not exacerbate even the cold sensitivity of this double mutant (data not shown). Therefore, the inability of PKC1 to act as a suppressor in the strain tif51A-1 zds1 zds2 is not likely the result of Pkc1 overactivation and toxicity. Thus, Zds1 may exert Pkc1-MAP kinase negative feedback and also act as a downstream member of a pathway leading to the Cdc42 effector Gic1. Furthermore, the influence of Zds1 and Zds2 over Cdc42 may not occur only as negative regulation. Two pieces of data support this idea: (1) high-copy ZDS1 is able to induce actin polarization in a tif51A-1 mutant at the restrictive temperature; and (2) decreasing Cdc42 activity through overexpression of its known GTPase-activating proteins (GAPs) Rga1 and Bem3 does not suppress the tif51A-1 mutant, suggesting that Zds1 is acting to promote suppression through a mechanism that does not involve Cdc42 inhibition.
It is important to note that high-copy CDC42 does not suppress the tif51A-1 mutant, but its function may be necessary for GIC1 suppression, since abolishing the Cdc42-Gic1 interaction, via CRIB domain mutations, also abolishes GIC1 suppression. This indication of the involvement of a specific Gic1 function in tif51A-1 suppression is reinforced by the fact that no other Cdc42 effectors were identified as suppressors of this eIF5A mutant. Also consistent with this observation, overexpression of Bni1, a formin proposed to act after and in a manner dependent on Gic1 (JAQUENOUD and PETER 2000), also suppresses the tif51A-1 temperature-sensitive phenotype.
Finally, we demonstrated that high levels of Gic1 and Bni1 can bypass the temperature-sensitive growth defect of the PKC1 mutant stt1, indicating that these factors may function downstream of Pkc1. On the other hand, ZDS1 does not suppress the stt1 mutant (data not shown), but this fact could be due to its proposed role in maintaining the balance of different signaling pathways, as mentioned above. Taken together, these data strongly support the existence of the proposed pathway linking Pkc1 to Gic1 through Zds1 and Zds2 (Figure 8) and connecting Pkc1 signaling to polarized growth.
Interestingly, another protein, Tos2, also connects Pkc1 to Cdc42, but via Cdc24 (DREES et al. 2001), and, recently, this protein has been shown to have a possible role in anchoring Cdc24 to the plasma membrane (TOENJES et al. 2004). Like Zds1 and Zds2, Tos2 contains multiple protein kinase C consensus phosphorylation sites and physically interacts with Pkc1 (DREES et al. 2001; TOENJES et al. 2004). Moreover, Skg6, which also interacts with both Zds1 and Zds2, shows interesting homology (35% identity, 48% similarity) to Tos2 (DREES et al. 2001; TOENJES et al. 2004). These protein interactions suggest a connection between Pkc1 and members of cell polarity determination in a common protein-protein network and further support the link between Pkc1 and Gic1 mediated by Zds1 and Zds2 as a novel pathway.
Curiously, here we also characterized SSD1 as a tif51A-1 suppressor in a low-copy plasmid. SSD1 is a polymorphic gene that encodes a protein that may or may not be functional, depending on the allele present in the genetic background of the yeast strain considered (STETTLER et al. 1993). Although its specific role has not been determined, a functional allele of SSD1 can suppress different mutants related to cell integrity and also the gic1 gic2 double mutant. Futhermore, ssd1 phenotypes can be suppressed by ZDS1 (TSUCHIYA et al. 1996; CHEN et al. 1997; KAEBERLEIN and GUARENTE 2002). Therefore, the isolation of SSD1 as a tif51A-1 suppressor strengthens the functional interaction between eIF5A and the Pkc1-Zds1-Gic1 pathway.
eIF5A function is important for actin cytoskeleton organization:
The identity of the eIF5A mutant suppressors described herein raised the hypothesis that a defect in establishment of cell polarity occurs in the tif51A-1 mutant. Subsequent analysis of the tif51A-1 strain actin cytoskeleton confirmed that this mutant shows defects in actin organization at the restrictive temperature. These data agree with a previous study, in which eIF5A was proposed to be important for translation of a subset of mRNAs involved in the G1/S transition, since depletion of this factor in yeast causes only a minor defect of total translation rate and an increase of enlarged cells with G1 morphology (KANG and HERSHEY 1994). The question of how eIF5A acts to assure correct polarized growth in S. cerevisiae is being investigated currently.
eIF5A is highly conserved throughout evolution, from archeabacteria to mammals, and this may reflect at some level a conservation of function. Therefore, as budding is not a mechanism ubiquitously used for eukaryotes to progress in the cell cycle, it would not be appropriate to propose a direct function for eIF5A in establishment of cell polarity in S. cerevisiae. Moreover, as mentioned before, overexpression of Pkc1, Zds1, and Gic1 cannot completely rescue eIF5A mutant defects, demonstrating that actin polarization is not the only function of this essential protein. Thus, it is unwise to assume that these proteins, including eIF5A, act directly in the same pathway. Conversely, eIF5A could control the expression of some factors important for G1/S transition such as the suppressors of the eIF5A mutant. Therefore, future studies involving the factors revealed herein may contribute to the elucidation of the role played by eIF5A toward specific gene expression.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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LITERATURE CITED |
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