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Vol. 17, Issue 3, 1164-1175, March 2006
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Department of Biochemistry, University of Geneva, CH-1211 Geneva 4, Switzerland;
* Department of Biochemistry, Biozentrum of the University of Basel, CH-4056 Basel, Switzerland;
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, CH-1211 Geneva 4, Switzerland; and
Graduate School of Biosphere Science, Faculty of Applied Biological Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
Submitted November 14, 2005;
Revised December 13, 2005;
Accepted December 20, 2005
Monitoring Editor: Sean Munro
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Murray et al., 2004). In the budding yeast Saccharomyces cerevisiae changes in transcription upon heat stress are fairly well understood, involving control by two transcription factors, the heat shock factor Hsf1p and Msn2p/4p. Hsf1p binds to heat shock elements (HSEs) found in the promoter region of many heat shock protein genes (Wu, 1995). Genes that do not contain HSEs, but whose transcription is induced by heat and other stress signals, including osmotic shock, DNA damage, and oxidative stress, contain stress response elements (STREs) in their promoters. On these stresses, Msn2/4p shuttles from the cytosol to the nucleus and activates transcription through binding the STREs (Schmitt and McEntee, 1996; Gorner et al., 1998). After transcription, the corresponding mRNAs are exported from the nucleus and are translated (Stutz and Rosbash, 1998). Proteins encoded by heat stress responsive genes are responsible for the synthesis of the thermoprotectant trehalose (Singer and Lindquist, 1998), for the folding of proteins and for the degradation of unfolded and aggregated proteins (Imai et al., 2003; Riezman, 2004).
In addition to the induction of heat shock proteins, yeast cells induce the de novo synthesis of free sphingoid bases, followed by ceramides and sphingolipids (Jenkins, 2003). The first steps in the biosynthesis of sphingolipids in animal cells and in yeast are similar, but differ in production of complex sphingolipids. In yeast, two sphingoid bases, sphinganine (commonly called dihydrosphingosine) and 4-hydroxysphinganine (commonly called phytosphingosine), can be converted upon addition of very-long-chain fatty acyl-CoA into ceramides. These ceramides are precursors for the three major classes of complex sphingolipids (Funato et al., 2002).
Interestingly, many of the cellular responses during heat stress depend on the up-regulation of sphingolipid synthesis (Jenkins, 2003) and yeast mutants unable to produce sphingolipids are hypersensitive to heat (Patton et al., 1992; Chung et al., 2000; Zanolari et al., 2000). One of these mutants carries a temperature-sensitive mutation in the LCB1 gene, called lcb1-100. The LCB1 gene encodes a subunit of the serine palmitoyl-transferase, which catalyzes the first step in sphingolipid synthesis (Buede et al., 1991). lcb1-100 mutants are therefore unable to produce sphingoid bases, ceramides, and sphingolipids during heat stress (Jenkins and Hannun, 2001). Addition of high concentrations of sphingoid bases to the growth media induces the synthesis of heat shock proteins at low temperatures (Dickson et al., 1997) and an lcb1-100 mutant was shown to be deficient in the synthesis of heat shock proteins (Friant et al., 2003). Mutant lcb1-100 cells also displayed specific transcriptional changes during heat stress (Cowart et al., 2003). This study showed that HSE- and STRE-dependent transcription does not depend greatly on the production of sphingoid bases.
Apart from sphingolipid synthesis, translation initiation is one of the key points for the regulation of gene expression and adaptation to stress (Dever, 2002). In eukaryotes, the small 40S ribosomal subunit interacts with the ternary complex composed of eIF2-GTP and the charged Met-tRNAiMet to form the 43S preinitiation complex, which then binds to the mRNA at the 5' end, scans for the initiator codon, and associates with the 60S ribosomal subunit to initiate translation (Kapp and Lorsch, 2004). Translation initiation can be regulated by various mechanisms including phosphorylation of the translation initiation factor eIF2
on serine 51 by the Gcn2p kinase, which down-regulates the overall translation initiation rate (Hinnebusch, 2000). The Gcn2p kinase is activated by several stimuli including membrane stress (Deloche et al., 2004).
The integrity of the 5'cap binding complex is also a target for general control of translation initiation for most cellular mRNAs (Gingras et al., 1999). One important regulator of cap-dependent translation initiation is eIF4G, which is degraded upon nutrient deprivation and in yeast cells deficient for YPK1/2 function (Berset et al., 1998; Gelperin et al., 2002). In addition, the eIF4E-binding proteins (4E-BPs) act as specific inhibitors. Binding of the 4E-BPs to eIF4E abolishes the interaction of eIF4E with eIF4G, thus blocking ribosome recruitment to the mRNA. Two functional homologues of mammalian 4E-BPs, Caf20p and Eap1p, have been described in S. cerevisiae but how these proteins regulate translation initiation is still poorly understood (Cosentino et al., 2000; Deloche et al., 2004).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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ade2 his3 leu2 trp1 ura3 can1 lag1::HIS3 lac1::ADE2 transformed with plag1–1TS::TRP1), eap1
(RH6178, Mata his3 his4 leu2 trp1 ura3 lys2 eap1::TRP1, this study), eap1lcb1-100 (RH6174, Mata his3 ura3 eap1 lcb1-100, this study), ypkts (YPT-40, Mat ypk1–1ts:HIS3 ypk2::TRP1 ade2 his3 leu2 lys2 trp1 ura3, kindly provided by J. Thorner), and mex67-5 (Mata ade2 his3 leu2 trp1 ura3 mex67:HIS3 [pUN100-LEU2-mex67-5], kindly provided by F. Stutz)
Previously described plasmids used in this study were as follows: pZJHSE2-137 containing an HSE, HSE2 from SSA1 promoter fused to LacZ, the pUKC414 vector containing the HSP26 promoter fused to LacZ (all described in Friant et al., 2003), plag1-1TS (Schorling et al., 2001), pAdh1-Msn2-GFP (Gorner et al., 1998), and pGM18/17 carrying a 7xSTRE-LacZ fusion for genomic integration (Marchler et al., 1993).
Polysome Analysis
All sucrose gradient analyses were performed exactly according to methods described (Foiani et al., 1991) except that sucrose gradients were 5–50% (wt/vol) and sedimentation was performed for either 2 h 45 min at 39 krpm or for 3 h 20 min at 35 krpm. Ribosomal subunit quantification was done in low-Mg2+ gradients as described (Foiani et al., 1991). All gradients were analyzed using an ISCO UV-6 gradient collector and continuously monitored at A254.
Western Blotting
Whole-cell extracts were prepared from 2 OD600 of cells grown in synthetic media at 24°C and shifted to 37°C for the indicated time as published (Deloche et al., 2004). For the analysis of eIF4G stability, equal amounts of protein from the different extracts were resolved by SDS-PAGE and subjected to Western blotting using a polyclonal antibody against eIF4G (kindly provided by M. Altmann). The blots were stripped and reprobed with polyclonal antibodies against Has1p (Emery et al., 2004).
Fluorescent In Situ Hybridization
Fluorescent in situ hybridization and analysis of total mRNA export was performed exactly as described (Cole et al., 2002). Analysis of HSP104 mRNA export was done using 10 ng of a mixture of oligos thj203 and thj205 modified with Cy3 following the protocol as described (Jensen et al., 2001). Samples were analyzed with a Zeiss Axioplan microscope (Thornwood, NY) using the appropriate filter sets.
Reverse Transcription-PCR
Total mRNA was isolated following the RNeasy protocol (Qiagen, Chatsworth, CA), and 2 µg of mRNA was analyzed using primers against lacZ mRNA (forward: 5'-CCCCGTTTACAGGGCGGCTTC, reverse: 5'-CCCCGTTTACAGGGCGGCTTC) and ACT1 (forward: 5'-CGGTTCTGGTATGTGTAAAGC, reverse: 5'-GGTGAACGATAGATGGACCAC) in a one-step reverse transcription reaction (Access RT-PCR System, Promega, Madison, WI). The reaction products were analyzed on 1.5% agarose gels, visualized, and quantified.
Translation Rate Assay
Translation rate measurements were adapted from Uesono and Toh (2002) and Deloche et al. (2004). In brief, 2 x 108 cells at early log phase were collected in 3 ml of synthetic dextrose media. Aliquots, 0.5 ml, were shifted to 37°C and labeled using [35S]methionine mix (Easy Tag EXPRESS-[35S] mix from New England Nuclear, Boston, MA) for 5 min up to the indicated time. Uptake and incorporation of [35S]methionine was stopped by 10 mM each NaN3 and NaF. One half of cells was mixed with an equal amount of 20% ice-cold TCA and incubated for 1 h on ice. The other half was resuspended in 10 more volumes of ice cold water, filtered using GF/C filters, and washed with ice-cold water. TCA precipitates were filtered using GF/C filters. The filtrate was washed using ice-cold 10% TCA and twice using ice-cold ethanol. All filters were dried and counted in a liquid scintillation counterusing a Packard Scintillation Counter (Packard Instrument Company, Downers Grove, IL).
Liquid 
-Galactosidase Assay
A liquid -galactosidase assay was performed as described previously with slight modifications (Miller, 1972). For each sample, time, OD420, and protein concentration, using a Bradford assay kit (Bio-Rad, Richmond, CA), was determined. The values reported are the average of at least three independent measurements.
Msn2 Green Fluorescent Protein
Cells expressing MSN2-green fluorescent protein (GFP) were grown to log phase at 24°C, shifted to 37°C for 10 min, and fixed for 2 h in phosphate-buffered saline-formaldehyde (3.7% final concentration) and analyzed essentially as described (Schmelzle et al., 2004).
Viability Assay
Log-phase cultures were grown in YPUAD at 24°C and an aliquot was shifted to 44°C. Samples were assessed for cell viability as described before (Friant et al., 2003). The viability experiments were repeated twice, yielding similar results.
UBI4 Suppression of the lcb1-100 Mutation
The RH3809 (lcb1-100) strain carrying a temperature-sensitive allele of the LCB1 gene was transformed with Yeplac181-UBI. UBI4 rescued growth at 37°C only in high copy number as previously reported (Friant et al., 2003).
Heat Shock Protein Labeling
Heat shock protein labeling was performed as described previously (Miller et al., 1979; Friant et al., 2003). Sphinganine isomers (Matreya, State College, PA) at 10 µM final concentration were added before the shift.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Because the promoters of both reporter constructs used in this study were shown to be dependent on Hsf1p (Stone and Craig, 1990; Amoros and Estruch, 2001), we repeated these assays using a stress-inducible promoter that contains seven artificially introduced STRE sequences and is solely dependent on Msn2/4p. Again -galactosidase activity of the 7xSTRE-lacZ reporter was greatly reduced in lcb1-100 cells compared with wild type during heat stress. This is in agreement with previously published data (Friant et al., 2003 and Figure 1A). The decrease in activity was not due to an increase in -galactosidase turnover as determined by pulse chase analysis (unpublished data). This shows again that induction of stress- and heat-inducible proteins is reduced in lcb1-100 cells.
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To elucidate how sphingolipid synthesis governs the production of heat shock proteins, we analyzed the nuclear import of the STRE-activating transcription factor Msn2p. Translocation upon heat stress of Msn2p to the nucleus was normal in lcb1-100 cells compared with wild type (Figure 1B). Therefore, cells defective for the synthesis of sphingolipids during heat stress are not impaired for the translocation of the stress-responsive transcription factor Msn2p to the nucleus.
We next assessed transcription of the 7xSTRE-lacZ, the SSA1-lacZ, and HSP26-lacZ mRNA in lcb1-100 cells. mRNAs for all three reporter constructs were up-regulated 30 and 60 min after heat stress in both wild-type and mutant cells. Induction of these reporter constructs was comparable in wild-type and lcb1-100 cells (Figure 1C and unpublished data). This result is in agreement with a recent study, showing that essentially none of the heat-induced heat shock mRNAs are affected in lcb1-100 cells after heat stress compared with wild type (Cowart et al., 2003).
Recent reports have shown the involvement of pre-mRNA splicing as a posttranscriptional regulatory mechanism during heat shock. In MOLT-4 cells, increased sphingolipid synthesis was shown to cause SR protein dephosphorylation (Jenkins et al., 2002), and SR proteins play a crucial role in splicing during heat stress (Shin et al., 2004). To analyze sphingolipid synthesis-dependent splicing during heat stress in yeast cells, we performed a primer extension of SNR17a exon 2. Wild-type as well as lcb1-100 cells were able to properly splice and join exons 1 and 2, resulting in an 81-base product, during up to 1 h of heat stress (Supplementary Figure S1). On the basis of these results we tend to exclude a role for sphingolipid synthesis-dependent splicing during heat stress in S. cerevisiae.
Export of Total and HSP104 mRNA Is Functional in lcb1-100 Cells
A reduced rate in heat shock protein synthesis was previously observed in cells defective for nuclear export of mRNA (Miller et al., 1979; Stutz and Rosbash, 1998). Consequently we analyzed both export of total and HSP104 mRNA from the nucleus during heat stress in wild-type, lcb1-100, and mex67-5 cells. For total mRNA we used a poly-dT probe that was labeled at its 3' end using digoxigenin (DIG)-modified dUTP. HSP104 mRNA was detected using oligos that hybridize specifically to this gene and were internally modified with Cy3 as described (Jensen et al., 2001). In wild-type and in lcb1-100 cells, total and HSP104 mRNA could be detected in the cytosol after 30 min of heat stress at 37°C, as seen by a diffuse cytosolic staining with no accumulation in the nucleus (Figure 2). As a control, we made use of the mex67-5 mutant. Mex67p is a mRNA export factor for a broad range of polymerase II transcripts, and mex67-5 cells accumulate total and heat-induced mRNA within the nucleus (Figure 2), as previously reported (Hurt et al., 2000). HSP104 mRNA staining in wild-type and lcb1-100 cells before heat stress was not detectable (unpublished data). We conclude that export of total and heat-induced mRNA is not affected in lcb1-100 mutant cells.
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; Uesono and Toh, 2002; Deloche et al., 2004). First, we determined the rate of protein synthesis in wild-type and lcb1-100 cells by comparing the amounts of [35S]methionine incorporation into protein during heat stress. Yeast cells were shifted from 24 to 37°C to induce heat stress and radiolabeled for 5 min as indicated (Figure 3A). Incorporation was stopped by adding azide and fluoride and the amount of 35S incorporated into protein was determined by TCA precipitation and scintillation counting.
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In contrast to wild-type cells the total uptake of [35S]methionine decreased in the lcb1-100 mutant cells during heat stress (unpublished data). Because the decrease in the TCA-precipitable counts in an aliquot of cells reflects both a decrease in uptake and incorporation into protein, the ratio of TCA-precipitated versus total cell-associated [35S]methionine for each aliquot was used to determine the rate of protein synthesis. The initial ratio of TCA-precipitable (incorporated into proteins) to total [35S]methionine (total cell associated) at 24°C (usually 
0.6) was set to 100%. Wild-type cells showed a transient decrease in protein synthesis after heat stress at 37°C but recovered and increased their rate of synthesis to 150% of the initial rate and maintained this for at least 1 h at 37°C. Mutant lcb1-100 cells showed the same decrease in protein synthesis upon heat stress, but there was no recovery. The rate of protein synthesis remained stable at 50% of the initial synthesis rate (Figure 3A).
To assess how sphingolipid synthesis affects translation during heat stress, we analyzed the sedimentation profiles of polysomes on linear 5–50% sucrose gradients. After the indicated times of heat stress, cycloheximide was added to the cultures to arrest translation elongation and to preserve the polysomes during preparation. Compared with wild-type cells, lcb1-100 already showed an increase in 80S monosomes, relative to 40S and 60S ribosomal subunits, at permissive temperature (Figure 3B). After 15 min of heat stress at 37°C, the sedimentation analyses showed that both cell types attenuated translation initiation as monitored by the increase in monosomes and the slight decrease in polysomes. The analyses of later time points revealed that this attenuation was transient for wild-type cells. In contrast, monosomes continued to increase in lcb1-100 cells. After 60 min at 37°C, wild-type cells returned to an almost normal distribution, but lcb1-100 cells showed a substantial defect in translation initiation as monitored by the large monosome peak with only very few actively translating polysomes (Figure 3B).
The samples from wild-type and lcb1-100 cells after 60 min of heat stress were then loaded onto 5–50% linear sucrose gradients containing 0.7 M NaCl. High salt concentration leads to the dissociation of randomly formed, inactive monosomes that are nontranslating and not tightly bound to mRNA (Foiani et al., 1991). In contrast to wild-type cells, the accumulated monosomes in lcb1-100 cells were nontranslating because almost the entire peak consisted of inactive 80S particles that could be dissociated into their 40S and 60S subunits (Figure 3C). Therefore, lcb1-100 mutant cells show the hallmarks of a translation initiation defect: a decrease in the polysome to monosome ratio and a majority of nontranslating monosomes.
To determine whether the synthesis of sphingolipids is required for translation initiation in general or only during heat stress, wild-type cells grown at 24 or 37°C were treated with the antifungal compound myriocin (ISP-1) that inhibits serine palmitoyltransferase (Horvath et al., 1994; Miyake et al., 1995). At 24°C the rate of protein synthesis remained stable at 100% for wild-type cells in the presence or absence of 10 µg/ml myriocin, whereas the same cells showed a decrease in the up-regulation of protein synthesis during heat stress in the presence of myriocin (unpublished data). Polysome distribution on sucrose gradients was normal for wild-type cells at 24°C after up to 2 h of treatment with myriocin (Figure 3D). In contrast, wild-type cells treated with myriocin at 37°C showed an increase in monosomes and a decrease in polysomes over time (Figure 3D). This demonstrates that sphingolipid synthesis is required for efficient translation initiation, mainly during heat stress. The effects of myriocin are weaker than those seen with the lcb1-100 mutant, but this is consistent with previous data (Horvath et al., 1994) and probably results from an incomplete inhibition of serine palmitoyltransferase by the compound.
The Synthesis of Sphingoid Base Is Required for Translation Initiation during Heat Shock
Metabolites of the sphingolipid biosynthesis pathway, including sphingoid base, ceramide, and sphingoid base-1-phosphate have been shown to be important second messengers in eukaryotic cells, regulating diverse biological processes such as cell growth, differentiation, apoptosis, stress responses, endocytosis, calcium homeostasis, and cell migration (reviewed in Dickson and Lester, 2002; Spiegel and Milstien, 2003). Our attention first focused on the phosphorylation of the sphingoid bases because accumulation of phosphorylated sphingoid bases resulted in increased thermotolerance and cell growth inhibition (Mandala et al., 1998; Kim et al., 2000). Yeast cells have two long-chain sphingoid base kinases, LCB4 and LCB5, which phosphorylate sphinganine and 4-hydroxysphinganine. Concomitant with the increase in sphingoid bases during heat stress, yeast cells show an increase in the amount of sphingoid base phosphates with a peak 15 min after heat stress (Ferguson-Yankey et al., 2002). Therefore we tested if LCB4 and LCB5 are required for the regulation of translation during heat stress.
Deletion of the sphingoid base kinases did not lead to a reduction in the production of heat shock proteins as measured by the induction of -galactosidase from SSA1 and HSP26 heat shock gene promoters (Figure 4A). Moreover, the rate of protein synthesis during heat stress was similar in wild-type and lcb4,5 double mutant cells. Interestingly, lcb4,5 cells did not show the typical, transient down-regulation in the rate of protein synthesis at 30 min of heat stress (Figure 4B). This indicates that accumulation of sphingoid bases or loss of sphingoid base phosphates, which was previously reported to occur in lcb4,5 cells (Ferguson-Yankey et al., 2002), increases the translation rate. The distribution of polysomes on sucrose gradients was analyzed from wild-type and lcb4,5 cells maintained at 24°C or after 15, 30, or 60 min of heat stress at 37°C, respectively. Polysomes remained unchanged in lcb4,5 cells as shown after 60 min of heat stress (Figure 4C). During early time points of heat stress wild-type and lcb4,5 cells showed the same distribution of polysomes (unpublished data).
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Sphingolipids and their precursor ceramide fulfill important functions in eukaryotic cells. Ceramide was shown to induce differentiation, cell cycle arrest in G0/G1 phase, senescence, and apoptosis, whereas sphingolipids have been implicated in cell-cell or ligand-receptor interactions, differentiation, and apoptosis in mammalian cells (Hannun and Obeid, 2002). Although ceramides and sphingolipids are up-regulated during heat stress in yeast, so far no direct involvement of these metabolites could be shown during this response. Therefore we analyzed the role of ceramide and complex sphingolipid synthesis in translation during heat stress.
Two homologous, redundant genes, LAG1 and LAC1, have been shown to be required for ceramide synthesis (Guillas et al., 2001; Schorling et al., 2001) and to be essential subunits of ceramide synthase (Vallee and Riezman, 2005). On deletion of these genes, yeast cells produce vastly reduced quantities of ceramides. Deletion of both genes leads to severe defects in cell viability and to decreases in heat stress resistance (Barz and Walter, 1999). To rule out the possibility that these phenotypes are of secondary nature and not directly connected to the loss of the de novo synthesis of ceramides, we introduced a temperature-sensitive allele of LAG1 on a plasmid in a strain deleted for both LAG1 and LAC1 (Schorling et al., 2001). The lac1lag1ts strain showed no defects in the induction of reporter constructs for heat shock proteins (Figure 4A). The rate of protein synthesis was also not affected in lac1lag1ts cells compared with wild-type cells (Figure 4B). In agreement with these results, the distribution of polysomes on sucrose gradients was similar in lac1lag1ts at 24°C and after heat stress at 37°C (Figure 4C) when compared with wild-type cells. To provide further evidence that ceramides are not required for regulation of protein synthesis after heat shock, we tested whether addition of the four different stereoisomers of sphinganine could complement the defects of lcb1-100 cells. Only two of these stereoisomers can be incorporated into ceramide (Watanabe et al., 2002). All four stereoisomers could restore normal regulation of heat shock protein synthesis, providing further proof that ceramide is not the sphingolipid required for regulation of translation (Figure 4D).
Phosphorylation of eIF2 Is Similar in Wild-Type and lcb1-100 Cells
Because a block in sphingolipid synthesis could possibly induce a membrane stress, leading to a decrease of translation initiation by activating the Gcn2p kinase, we next monitored the phosphorylation status of eIF2 (encoded by SUI2 in yeast) on serine 51 during heat stress in wild-type and lcb1-100 cells. Western blots against total and phosphorylated eIF2 were performed. Wild-type and lcb1-100 cells showed an increase in phosphorylation of eIF2 shortly after shift to 37°C. During prolonged times of heat stress, decreased amounts of phosphorylated eIF2 were observed in lcb1-100 cells compared with wild-type cells (Supplementary Figure S2). The lack of difference at 15 and 30 min allow us to exclude a role of the Gcn2p-eIF2 pathway for regulation of heat shock protein translation in our mutant cells, but it could play some role in the recovery process.
Deletion of the Yeast eIF4E-binding Protein, Eap1p, Restores Translation Initiation and Synthesis of Heat Shock Proteins
A second control step in translation initiation is achieved by the control of the availability of eIF4E proteins and deletion of EAP1 was shown to partially restore translation initiation of membrane-stressed cells (Deloche et al., 2004). Therefore, we analyzed the rate of protein synthesis in eap1, and eap1D lcb1-100 cells. Deletion of EAP1 alone had no effect on the rate of protein synthesis during heat stress (Figure 5A). In contrast, deletion of EAP1 in lcb1-100 cells was able to restore the incorporation of [35S]methionine into protein to almost wild-type levels. Interestingly, similarly to lcb4,5 cells, eap1lcb1-100 cells showed no transient down-regulation in the synthesis rate shortly after heat stress (Figure 5B). Deletion of CAF20 in wild-type, lcb1-100, eap1, or eap1lcb1-100 cells did not lead to any phenotypes different from presented above (unpublished data).
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Polysome analyses of eap1, lcb1-100, and eap1lcb1-100 cells demonstrated that eap1 cells behave as wild-type cells before and after 60 min of heat stress at 37°C (Figure 5B). As before, mutant lcb1-100 cells showed an accumulation of monosomes (80S peak) at permissive temperature and strong accumulation of nontranslating monosomes after 60 min of heat stress. In contrast, eap1lcb1-100 cells showed a less-severe loss of translating ribosomes (Figure 5B). Polysome to monosome ratio (p/m) was analyzed and was around 4.7 for wild-type and eap1 cells before and after heat stress (SD < 10%). lcb1-100 and eap1 lcb1-100 already displayed a p/m ratio of 1.75 before heat stress. After 60 min at 37°C, the p/m ratio of lcb1-100 cells shifted dramatically to 0.18, whereas eap1lcb1-100 cells showed a significant higher p/m ratio of 0.68. The increase in the protein synthesis rate at early time points during heat stress was not evident as a change in the distribution of polysomes or in the size of the 80S peak in eap1lcb1-100 cells, as observed for the lcb4,5 mutant (unpublished data).
Next we determined if deletion of EAP1 restored synthesis of heat shock proteins to lcb1-100 cells. Induction of SSA1 and HSP26 heat shock gene promoters fused to -galactosidase was increased in eap1 cells compared with wild-type cells, especially for the HSP26 fusion, and eap1 restored heat shock protein induction to lcb1-100 cells (Figure 5C).
If loss of production of heat shock proteins is the major cause of decreased viability during heat stress in cells deficient for sphingolipid synthesis, eap1 lcb1-100 cells would be predicted to have improved viability due to the restoration of heat shock protein synthesis. Therefore, we assayed resistance to heat shock at an elevated temperature. Wild type, lcb1-100, eap1, and eap1lcb1-100 cells were heat shocked at 44°C and the percentage of cells able to form colonies at permissive temperature was determined as a function of time. The lcb1-100 mutant showed a strong defect in survival at high temperature. In contrast, lcb1-100 eap1 cells had a greater resistance to heat treatment over the 2-h period (Figure 5D).
Taken together these results show that deletion of the translation repressing 4E-BP Eap1p increased translation of heat shock proteins in lcb1-100 cells. This indicates that synthesis of sphingoid bases during heat stress regulates translation initiation at a cap-dependent step. In addition, these results show that recovery of heat shock protein synthesis allows lcb1-100 cells to resist heat shock in the absence of sphingolipid synthesis. Therefore, the essential function of sphingolipids in resistance to heat shock is their function in heat shock protein production.
Translation Initiation Is Regulated in Part via the Conserved PKH/YPK Signaling Cascade
Sphingoid bases were previously shown to serve as signaling molecules. Addition of sphingoid base stimulated phosphorylation of the yeast serum- and glucocorticoid-inducible kinase (SKG) homologues Ypk1p and Ypk2p by the upstream yeast PDK1 homologues Pkh1p and Pkh2p (Casamayor et al., 1999) in an in vitro assay (Friant et al., 2001). Also overexpression of YPK1 conferred resistance to the serine palmitoyltransferase inhibitor myriocin, (Sun et al., 2000). Furthermore, it was shown that cells thermosensitive for YPK signaling are defective for translation initiation and that the translation initiation factor eIF4G is depleted with time at nonpermissive temperature (Gelperin et al., 2002). If the proposed conserved signaling cascade is operative here, eIF4G, as a readout, should be depleted in lcb1-100 at similar rates during heat stress as was observed for a ypkts strain. To test this, Western blots against eIF4G were performed.
eIF4G was stable in wild-type cells up to 4 h during heat stress at 37°C. In contrast, lcb1-100 cells showed loss of eIF4G beginning at 60 min of heat stress and after 4 h only small levels of eIF4G could be detected. Has1p, a member of the DEAD-box family of RNA helicases that is involved in 40S ribosomal subunit biogenesis (Emery et al., 2004), remained stable in both wild-type and lcb1-100 cells at all times tested (Figure 6A). At the time, it is not known how stability of eIF4G is controlled. However, our results demonstrate clearly that the stability of eIF4G in lcb1-100 cells is comparable to ypkts cells, which suggests a similar mechanism of regulation.
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Because the arrest in translation initiation in pkhts cells was rather slow compared with lcb1-100 cells, we wanted to know if induction of SSA1 and HSP26 heat shock gene promoters fused to -galactosidase was affected in pkhts and ypkts cells. Although pkhts cells showed a nearly 50% reduction in the induction of SSA1-lacZ and HSP26-lacZ, ypkts cells displayed a induction profile similar to wild-type cells (Figure 6D). These results suggest that translation is regulated by the sphingoid base-dependent PKH kinase during heat stress and that the Pkh kinases may have other targets than the Ypk kinases in this pathway.
Ubiquitin Overexpression Can Partially Suppress the Translation Defect in lcb1-100 Cells
Overexpression of the polyubiquitin gene UBI4 restored growth of lcb1-100 cells at elevated temperatures without restoring heat shock protein expression. UBI4 overexpression led to higher levels of ubiquitin and enhanced degradation of un- or misfolded proteins in a proteasome-dependent pathway, preventing aggregation of those proteins (Friant et al., 2003). Overexpression of ubiquitin partially suppressed the decrease in the translation rate as determined by measuring the incorporation of [35S]methionine into proteins (Figure 7A). Mutant lcb1-100 cells overexpressing UBI4 showed the same distribution of polysomes on sucrose gradients as wild-type cells at permissive temperature. After 60 min of heat stress, lcb1-100 cells overexpressing ubiquitin showed fewer accumulated monosomes and more polysomes compared with the polysomes of lcb1-100 cells shown in Figure 3B (Figure 7B). This was in agreement with the translation rates measured. Therefore, the overexpression of ubiquitin in the lcb1-100 mutant largely abrogates the need of sphingoid bases and heat shock proteins for the recovery of protein synthesis after a heat stress.
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DISCUSSION |
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). These results could have implicated sphingoid bases in the induction of the heat stress response, in particular in mRNA induction, or could have meant that high amounts of sphingoid bases induce a stress themselves, in particular because high amounts of sphingoid bases are toxic to yeast cells (Mao et al., 1999). The role of sphingolipid biosynthesis in heat shock protein expression was then confirmed using the lcb1-100 mutant (Friant et al., 2003). Interestingly, transcription of most heat shock protein genes was not dependent on sphingolipid biosynthesis (Cowart et al., 2003), which already suggested that the level of control was not transcriptional. The results presented here demonstrate that synthesis of sphingoid base phosphates, ceramides, and sphingolipids are not required for translation and synthesis of heat shock proteins during a heat shock. This result is both interesting and intriguing because all metabolites of the sphingolipid biosynthesis pathway were shown to be up-regulated during heat stress. A convincing role for sphingoid base phosphates, ceramides, and complex sphingolipid synthesis in the heat stress response in yeast remains to be shown. Alternatively, they could serve as sinks to remove sphingoid bases once they have performed their signaling function.
Heat stress leads to a rapid but temporary repression of ribosomal protein (RP) gene transcription in wild-type cells (Gasch et al., 2000). In contrast, RP mRNA levels failed to be down-regulated during heat stress in lcb1-100 cells (Cowart et al., 2003 and Supplementary Figure S3A). In addition, total 25S and 18S rRNA seemed to be reduced in lcb1-100 cells in the absence and at the early stages of heat stress (Supplementary Figure S3B). However, this is unlikely to affect translation by the availability of ribosomes because RP gene mRNA was not reduced to an amount below that of wild-type cells during heat stress. Quantification of ribosomal 40S and 60S subunits using low Mg2+ extracts gave a ratio of 1.6 (60S:40S) for wild-type and lcb1-100 cells at 24°C and after 60 min of heat stress at 37°C, respectively (unpublished data). Current data also indicate that there is a sphingolipid-dependent regulation of tRNA transcript levels in response to heat (Cowart et al., 2003), although mechanisms regulating tRNA levels in yeast are unknown. Despite this, loading of tRNA with amino acids did not seem to be affected because phosphorylation of eIF2 was not increased compared with wild-type (Supplementary Figure S2).
Surprisingly, the defect in sphingolipid synthesis did not elicit a membrane stress that is typically witnessed by strong phosphorylation of eIF2 (Deloche et al., 2004). Nevertheless, deletion of the 4E-BP EAP1 partially restored heat shock protein and general translation in lcb1-100 mutants in a manner similar to cells showing a transient attenuation in translation initiation resulting from membrane stress. This result, together with the fact that eIF4G is depleted in lcb1-100 cells, indicates that sphingoid bases regulate translation initiation through a cap-dependent step. Growth at high temperatures could not be restored by an EAP1 deletion in lcb1-100, but this was to be expected because deletion of EAP1 alone causes defects in genetic stability at 37°C (Chial et al., 2000). We do not know if this or the hyperactivation of the stress response is the primary cause for lethality in eap1 cells. At this stage we cannot distinguish between sphingoid bases signaling directly to Eap1p or regulation of a parallel pathway. The precise role of Eap1p still needs to be determined.
Our data suggest that the defect in heat shock protein translation is most likely due to a reduced activity of the sphingoid base-dependent PKH/YPK signaling pathway. A mutant defective for YPK kinases showed a strong decrease in translation initiation during prolonged heat stress and depletion of eIF4G (Gelperin et al., 2002) was observed at similar rates in lcb1-100 cells. Mutant cells defective for PKH1/2 or YPK1/2 signaling however showed strong defects in the rate of heat shock protein induction for the former and almost no defect for the latter. This suggests that the roles of the kinases are probably different in the two phases of response to heat shock. The first phase regulates the immediate translation of heat shock mRNAs, requires sphingoid bases, and is strongly dependent on Phk kinases, but independent of Ypk kinases. The reason why the pkh-ts mutant phenotype is weaker than that of the lcb1-100 mutant is not known, but it could be that the Pkh kinase encoded by the ts allele is not immediately inactivated at 37°C. Alternatively, it could be that the Pkh kinases are not the only vehicles mediating the sphingoid base regulation of heat shock protein expression.
The second phase is the increase in the rate of translation in general. This phase is likely to depend on the function of heat shock proteins in the first phase because overexpression of ubiquitin partially restored the translation recovery defect in lcb1-100 cells. Ubiquitin has been shown to functionally substitute for heat shock proteins under these conditions and its role must be in the recovery phase because its overexpression does not restore heat shock protein induction in lcb1-100 cells (Friant et al., 2003). One way to rationalize these results is that sphingoid bases are required for heat shock protein translation and that in the normal course of events, heat shock proteins play a crucial role in the recovery process. However, in the absence of heat shock proteins, protein aggregation or misfolded proteins could have an additional negative effect on translation preventing translation recovery. This latter effect could be circumvented by the overexpression of ubiquitin. It has been shown that the yeast Hsp70 homologue Ssa is required for efficient translation by promoting the interaction of Pab1p with eIF4G. Depletion of Ssa led to reduced translation and reduction of eIF4G even in the absence of heat stress (Horton et al., 2001). Therefore, accumulation of unfolded proteins and/or aggregates due to an inefficient heat shock protein induction could lead to a titrating out of Ssa proteins and a block in translation initiation. Overexpression of ubiquitin could remove the unfolded/aggregated proteins thus making the limiting amount of Ssa available for its function in translation. Sphingolipid synthesis may also play a role in the recovery process because eIF4G is depleted in lcb1-100 cells similarly to the reduction found in ypkts mutants and the Ypk kinases are part of a sphingoid base activated protein kinase cascade in yeast (Casamayor et al., 1999; Friant et al., 2001)
Sphingolipids as a General Signal for Heat Stress
Although we cannot absolutely rule out an indirect role of sphingoid base synthesis in gene expression, we prefer the hypothesis of a direct role for sphingoid bases in translation initiation during recovery from heat stress. The results presented here and previously demonstrate that the synthesis of sphingoid bases act as a general signal for the cellular responses to heat stress. To survive a heat stress, cells need to change their transcriptional program and the newly transcribed messages need to be processed and translated efficiently in order to change the developmental program of the cell (Preiss et al., 2003). During this time, the cell cycle is arrested in order to give the cell enough time to make these changes and to rearrange its actin cytoskeleton (Delley and Hall, 1999). In cells lacking sphingolipid synthesis, ribosomal proteins were found to be less down-regulated and translation initiation failed to take place efficiently. In addition, cells deficient for sphingolipid synthesis fail to arrest in the cell cycle (Jenkins and Hannun, 2001) and cannot reorganize their actin cytoskeleton (Friant et al., 2000). The trigger for these responses is probably the increase in de novo sphingolipid biosynthesis, which is extremely fast and most likely controlled at the first committed step, serine palmitoyltransferase. Most studies of the regulation of this enzyme indicate that its activity is controlled by the availability of its substrates, serine and palmitoyl-CoA (Merrill et al., 1988). One of the first events that ensue during a heat shock is a reduction in the rate of protein synthesis, which logically would lead to an increase in available serine, and could provide an explanation for the reason why sphingoid bases have evolved to regulate the heat shock response. Serine palmitoyltransferase activity also increases upon heat shock in mammalian cells (Jenkins, 2003).
Sphingolipid synthesis could therefore act as a sensor for heat stress, coupling an essential metabolic process to a diverse set of cellular responses like transcription, translation, cell cycle progression, actin organization, secretion, and endocytosis. To further understand the responses to stress in connection to sphingolipid synthesis is of particular interest for many fields in biology and medicine. Recently it was shown that dihydromotuporamine C (dhMotC), a compound that inhibits angiogenesis and metastasis, targets the sphingolipid pathway (Baetz et al., 2004) and therapeutic radiation stimulates the synthesis of ceramide in tumors (Santana et al., 1996). Understanding the molecular functions of the sphingolipid synthesis intermediates would greatly facilitate our understanding of disease states and therapeutic methods.
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ACKNOWLEDGMENTS |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Howard Riezman (howard.riezman{at}biochem.unige.ch).
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