Protein Kinase C Enables the Regulatory Circuit That Connects Membrane Synthesis to Ribosome Synthesis in Saccharomyces cerevisiae*

Concepcion R. NierrasDagger and Jonathan R. Warner§

From the Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The balanced growth of a cell requires the integration of major systems such as DNA replication, membrane biosynthesis, and ribosome formation. An example of such integration is evident from our recent finding that, in Saccharomyces cerevisiae, any failure in the secretory pathway leads to severe repression of transcription of both rRNA and ribosomal protein genes. We have attempted to determine the regulatory circuit(s) that connects the secretory pathway with the transcription of ribosomal genes. Experiments show that repression does not occur through the circuit that responds to misfolded proteins in the endoplasmic reticulum, nor does it occur through circuits known to regulate ribosome synthesis, e.g. the stringent response, or the cAMP pathway. Rather, it appears to depend on a stress response at the plasma membrane that is transduced through protein kinase C (PKC). Deletion of PKC1 relieves the repression of both ribosomal protein and rRNA genes that occurs in response to a defect in the secretory pathway. We propose that failure of the secretory pathway prevents the synthesis of new plasma membrane. As protein synthesis continues, stress develops in the plasma membrane. This stress is monitored by Pkc1p, which initiates a signal transduction pathway that leads to repression of transcription of the rRNA and ribosomal protein genes. The importance of the transcription of the 137 ribosomal protein genes to the economy of the cell is apparent from the existence of at least three distinct pathways that can effect the repression of this set of genes.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The cell has evolved complex mechanisms to reproduce its major components, e.g. its genome, its translational apparatus, and its complement of membranes, including most particularly the plasma membrane. In recent years there have been great strides in understanding the biochemistry and physiology of these systems. Yet we know very little about how they are coordinated, about how the cell manages to maintain balanced output of ribosomes, nuclear envelope, and plasma membrane, to name just a few.

In a search for elements regulating the synthesis of ribosomes in Saccharomyces cerevisiae we found an unexpected connection between two of these mechanisms (1, 2). Namely, any failure in the secretory pathway, caused by mutants in components of the early (sec61 and sec63) or late (sec53) endoplasmic reticulum (ER),1 of the ER-Golgi complex (sly1 and sec18) or of the trans-Golgi network (sec7, sec14, and sec1) leads to a nearly complete repression of the transcription of the genes encoding the components of the ribosome, both those for ribosomal proteins (RPs) and those for ribosomal RNA. Drugs that inhibit different steps of the secretory pathway, such as tunicamycin and brefeldin A, lead to a similar repression of ribosomal genes (1).

In S. cerevisiae ribosome synthesis accounts for a major portion of the biosynthetic capacity of the cell. We estimate that rRNA transcription represents 60% or more of the total transcription of the cell (3). mRNAs encoding RPs represent >4400 of the 15,000 mRNAs of the cell (4, 5). Because they have relatively short half-lives,2 we estimate that transcription of RP genes accounts for ~50% of the initiation events by RNA polymerase II. Furthermore, ribosome synthesis involves the coordination of ~100 rRNA genes and 137 RP genes encoding 78 different RPs (7) to provide equimolar amounts of each component (3, 8, 9). Finally, this coordinate synthesis is responsive to wide variety of environmental stimuli, both positive and negative, such as the dynamics of growth in culture (10), changes in carbon source (11, 12), amino acid starvation (13, 14), and manipulation of the cAMP-protein kinase A pathway (15, 16).

In what way could the secretory pathway be connected to the transcriptional machinery for ribosome synthesis? In yeast, relatively few proteins are secreted, and those are secreted mostly to participate in the construction of the cell wall. The main function of the "secretory pathway" is to construct new membranes. Therefore, we suggest that the repression of the RP genes in response to a defect in the secretory pathway is one manifestation of an intracellular signal transduction pathway that serves to maintain balanced growth of the several components of the cell. If the secretory pathway fails, it is to the advantage of the cell to cease its substantial investment in new ribosomes.

We have explored the possible participation of a variety of signal transduction pathways in the regulatory circuit that links membrane synthesis with ribosome synthesis. Neither the unfolded protein response (UPR) known to connect the ER and transcription (17), nor the stringent response that represses ribosome synthesis in response to an insufficient supply of amino acids (13, 14), nor the protein kinase A pathway, which appears to regulate ribosome biosynthesis in response to carbon source, through RAS and cAMP, (16, 18) appear to be involved. Rather, our results suggest that the primary causal event is stress on the plasma membrane, because of continued protein synthesis in the absence of new membrane formation. Protein kinase C (PKC), known to monitor the integrity of the plasma membrane (19, 20), participates in transducing the signal between the plasma membrane and the transcriptional apparatus.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Strains and Genetic Methods-- The yeast strains used in this work are listed in Table I. Strains were constructed by crosses and tetrad dissection following standard yeast methods (21).

An ire1Delta ::LEU2 disruption cassette was constructed as follows: 600 bp from each of the 5' (nucleotides 12-587) and 3' (nucleotides 3276-3868) coding regions of IRE1 were amplified by polymerase chain reaction from genomic DNA from W303 using primers derived from the gene sequence in the data base. For ease of cloning, the 5' fragment was flanked by engineered SacI and XbaI sites, and the 3' fragment was flanked by engineered EcoRI and SalI sites. The fragments were cloned into the multiple cloning site of pBluescript (Stratagene). The yeast LEU2 gene was isolated as a 2.2-kb XbaI-PstI fragment from pUC-LEU2 and inserted between the IRE1 sequences. The ire1Delta ::LEU2 fragment was isolated by digestion with SacI and XhoI, and nonhomologous flanking sequences were removed by digestion with Bal31 nuclease. W303 was transformed using lithium acetate (22). The MN5 strain requires media supplemented with inositol (23). The disruption of IRE1 was confirmed by Southern blot analysis.

Congenic MN13, MN14, MN15, and MN16 strains were isolated from a cross of S13-7C with 312. Disruption of BCY1 was confirmed by testing thermotolerance (24) of the relevant strains (data not shown).

Congenic MN51, MN52, MN53, and MN54 strains were isolated from a cross between NY3 and DL377. The LEU2 marker could not be used to follow pkc1Delta , and the deletion was confirmed by testing for the requirement of pkc1Delta strains for osmotic support (1 M sorbitol) (25).

Plasmids-- Strains were transformed either with the CEN- and URA3-based vector YCp50 (26), or with YCp50 containing Ras2Val-19 in the multiple cloning site (YCpR2V). YCpR2V was a gift from Tamar Michaeli.

Growth Conditions-- Except for MN51-MN54, strains were typically maintained on YPD plates (2% yeast extract, 1% Bacto-peptone, 2% glucose, and 2% agar) at 23 °C. MN51-MN54 strains were maintained on YPD plates supplemented with 1 M sorbitol. For temperature shift experiments, strains were grown overnight in the indicated media at 23 °C to early log phase, 1-1.5 × 107 cells, and then shifted to 37 oC by transfer to a large, prewarmed flask. Aliquots were taken at specified times, and total RNA was prepared.

Tunicamycin was from Sigma and was stored as a 5 mg/ml stock solution in 75% methanol at -20 oC. 3-Amino-1,2,4-triazole from Sigma was stored as a 0.5 M stock solution in water at 4o. Chlorpromazine (CPZ) from Sigma was stored as a stock concentration of 25 mM.

Liquid stock cultures grown in YPD (plus 1 M sorbitol for MN51-MN54) were used to seed cultures for labeling experiments. Labeling experiments were done in synthetic glucose medium minus methionine (21), plus 1 M sorbitol for MN51-MN54.

RNA Isolation and Northern Blot Analysis-- Total RNA was prepared from harvested cell samples by a modification of the method of Schmitt et al. (27). The cells were disrupted by agitation with sterile glass beads in 400 µl of AE buffer (50 mM Na acetate, pH 5.3, 10 mM EDTA) plus 40 µl of 10% SDS. Recovered RNA was typically resuspended in 30 µl of sterile water and stored frozen at -70 °C. RNA was run on 1.5% agarose gels with formaldehyde, as described previously (1). For Northern blot analysis, RNA was blotted to Nytran nylon membrane. 32P-Labeled antisense RNA probes were used for RPL30,3 RPL3, and ACT1. 32P-Labeled DNA probes for KAR2, HIS4, and ARG3 were prepared by random primer extension of polymerase chain reaction-amplified fragments. Oligonucleotide probes for RPS28, RPL8, and U3 small nucleolar RNA were labeled with 32P using polynucleotide kinase. ACT1 and U3 were used as independent loading standards. RNA levels were quantified with respect to the internal controls using PhosphorImager (Molecular Dynamics) analysis.

Labeling Experiments-- Transcription and processing of rRNA were detected by post-transcriptional incorporation of methyl groups (28;29). For pulse-chase experiments, strains were grown at 23 oC to log phase in medium minus Met (plus1 M sorbitol when needed) (21). An aliquot of each culture was pulse-labeled with [C3H3]methionine (60 µCi/ml; DuPont NEN) at room temperature for 3 min before chasing with nonradioactive methionine (500 µg/ml). Samples were taken by pouring onto crushed sterile ice. Cells were harvested by centrifugation and were stored at -70 °C. RNA was prepared and analyzed on agarose gels as described above.

To visualize the C3H3-labeled RNA, agarose gels were soaked in En3Hance (DuPont NEN), washed in water, dried, and visualized by radiography.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In trying to establish the mechanisms by which a failure in the secretory pathway represses the transcription of the components of the ribosome, there are three general areas to consider: known mechanisms by which aspects of the secretory pathway influence transcription, known mechanisms that govern ribosome synthesis, and known mechanisms by which membrane stress influences transcription.

The Repression of RP Genes Is Not Dependent on IRE1-- The most thoroughly understood relationship between the secretory pathway and the regulation of gene expression is the UPR (for review, see Refs. 30 and 31), in which the accumulation of misfolded or unfolded proteins within the ER is sensed by the transmembrane kinase Ire1p. Activation of Ire1p kinase effects the production of alternatively spliced mRNA encoding the transcription factor Hac1p (32, 33). Hac1p then activates the transcription of the set of UPR-responsive genes (31), including the chaperonin, KAR2, the yeast homolog of mammalian BiP (34, 35). Induction of KAR2 therefore requires the presence of IRE1 (36-38).

To ask whether the repression of RP gene transcription was related to the UPR, we attempted to delete the IRE1 gene in our wild type and sly1-1 strains. However, sporulation of an IRE1/ire1Delta SLY1/sly1-1 diploid led to no viable ire1Delta sly1-1 spores from the dissection of 38 tetrads. Therefore, we conclude that deletion of IRE1 in a sly1-1 background is lethal. The presence of the sly1-1 allele leads to modest induction of KAR2 even at permissive temperatures (data not shown). Sly1p acts at the level of the ER and Golgi (39, 40). Perhaps even at the permissive temperature the sly1-1 mutation leads to sufficient disruption of the ER and Golgi that some Kar2p activity is essential to maintain viability.

As an alternative, the wild-type strain carrying ire1Delta was treated with tunicamycin, which inhibits protein glycosylation within the ER, activating the UPR (37). This leads to a strong induction of KAR2 (Fig. 1, left), which does not occur in the absence of a functional IRE1 gene (Fig. 1, right). ACT1 transcripts and U3 small nucleolar RNA, used as loading controls, are not affected. The blot was then probed with genes encoding two ribosomal proteins, RPL3 and RPL30. RPL30 was chosen to represent the large majority of RP genes with transcription that is activated by the binding of Rap1p (28, 41, 42); RPL3 represents a minority of RP genes with transcription that is activated by the binding of Abf1p (43). Each gene is repressed in the presence of tunicamycin (Ref. 1 and Fig. 1, left), even in the absence of IRE1 (Fig. 1, right). Because the repression of RP gene transcription does not require Ire1p, it is not a manifestation of the UPR.


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Fig. 1.   The IRE1 gene is not involved in the repression of RP gene transcription imposed by a secretion defect. Northern blot showing response of ire1Delta strain to tunicamycin. RNA samples were prepared from separate experiments. Strains W303 and MN5, isogenic except at IRE1, were grown overnight in YPD at 23 °C and then treated with tunicamycin at a final concentration of 2.5 µgm/ml. Total RNA was prepared and analyzed by Northern blot as described, using radiolabeled probes for RPL30, RPL3, ACT1, KAR2, or U3 as described under "Materials and Methods." 10 µg of total RNA was loaded in each lane.

The Repression of RP Genes Is Not a Classic Stringent Response-- The classic case of coordinate repression of rRNA and ribosomal protein syntheses is the stringent response in Escherichia coli (for review, see Ref. 44). In cells deprived of an essential amino acid, protein synthesis declines and transcription of rRNA is repressed, probably by the accumulation of the hyperphosphorylated forms of guanosine, ppGpp and pppGpp. This leads to a subsequent inhibition of ribosomal protein synthesis, largely but not entirely mediated through translational control. Transcription of other genes, generally those encoding biosynthetic enzymes, is coordinately induced.

S. cerevisiae deprived of an amino acid responds by repressing the transcription of both rRNA and RP genes (13), a stringent response by analogy to the phenomenon observed in E. coli. An important corollary of the stringent response is the induction of a number of genes involved in amino acid biosynthetic pathways, such as HIS4 and ARG3 (14). Treating S. cerevisiae with the antimetabolite 3-amino-1,2,4-triazole mimics starvation for histidine, also triggering a stringent response, as seen in Fig. 2, lanes 5-8, where HIS4 and ARG3 are strongly induced, and the two RP genes are repressed.


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Fig. 2.   The repression of RP gene transcription by a secretion defect is not a starvation response. Strain 312, sly1-1, was grown overnight in supplemented minimal media at 23 °C and then either shifted to 37 °C or treated with 10 mM 3-amino-1,2,4-triazole at 23 °C. Total RNA was isolated and analyzed by Northern blot using radiolabeled probes for ACT1, HIS4, ARG3, RPL30, RPL3, or U3. 5 µg of total RNA was loaded in each lane.

Is the repression of RP mRNA transcription in response to a defect in the secretory pathway a manifestation of the stringent response? Disrupting the secretory pathway by shifting a sly1-1 strain to the nonpermissive temperature leads to the usual repression of the RP genes (Fig. 2, lanes 1-4), but there is no concomitant induction of HIS4 or ARG3. Therefore, we conclude that the repression of RP gene transcription attributable to a defect in the secretory pathway is not a manifestation of the stringent response.

The Repression of RP Genes Is Not Affected by Activation of Protein Kinase A-- The transcription of ribosomal components is exquisitely sensitive to growth conditions, such as nitrogen availability and carbon source (12, 45, 46). Extensive experiments have shown that addition of glucose leads to signal transduction through the RAS pathway to stimulate adenylyl cyclase and to activate cAMP-dependent protein kinase (47, 48). Activation of the RAS-cAMP-protein kinase A pathway, either by addition of cAMP (18), or by deletion of BCY1, the regulatory subunit of protein kinase A (16), leads to increased transcription of RP genes. If the protein kinase A pathway were responsible for mediating between the secretory pathway and ribosome synthesis, a constitutively activated protein kinase A pathway should prevent the repression of RP gene transcription in response to a defect in the secretory pathway.

Strains with a constitutively activated cAMP pathway were constructed in two ways. First, a multicopy plasmid expressing the Ras2Val-19 form of RAS2 (49) was introduced into strains that were wild type, sly1, sec1, or sec63. This mutation attenuates the Ras2p GTPase activity, leading to higher concentrations of cAMP, and to other manifestations of the activated protein kinase A response, such as increased sensitivity to heat shock (49, 50). Each of the transformed strains was found to have increased sensitivity to a heat shock at 55 °C (data not shown). When sly1-1 or sec1-1 (Fig. 3) or sec63 cells (not shown) were shifted to the nonpermissive temperature, there was in each case a repression of transcription of RP genes that was barely, if at all, affected by the presence of the Ras2Val-19 gene (Fig. 3A, compare lanes 9-12 with lanes 13-16 and 17-20).


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Fig. 3.   The repression of RP gene transcription by a secretion defect is not affected by Ras. A, Northern analysis of wild-type or secretory mutants transformed with vector (+YCp50) or with plasmid carrying RAS2Val-19 (+YCpR2V). Cultures were grown overnight in media without uracil at 23 °C and then shifted to 37 °C. Total RNA was isolated and analyzed by Northern blot using radiolabeled probes for ACT1, RPL3, RPL30, or U3. 5 µg of total RNA was loaded in each lane. B, Northern analysis of isogenic strains subjected to heat shock. Cultures were grown overnight in YPD at 23 °C and then shifted to 37 °C. Total RNA was isolated and analyzed by Northern blot using radiolabeled probes for ACT1, RPL3, RPL30, or U3. 5 µg of total RNA was loaded in each lane. The genotypes listed above the lanes represent the following strains (see Table I): SLY1 BCY1, MN15; SLY1 bcy1Delta , MN16; sly1-1 BCY1, MN14; and sly1-1 bcy1Delta , MN13.

An alternate way to constitutively activate the cAMP pathway is by inactivation of Bcy1p, the regulatory subunit of protein kinase A. Deletion of BCY1 leads to constitutively high levels of protein kinase A activity (24, 51). However, as shown in Fig. 3B, deletion of BCY1 does not alleviate the repression of RP gene transcription in response to a defect in the secretory pathway (compare lanes 9-12 with 13-16). Thus we conclude that a failure in the secretory system represses ribosome synthesis in some way other than the cAMP pathway.

In wild-type cells there is a temporary reduction in the level of RP mRNA in response to heat shock (52) (Fig. 3, A and B, lane 2). This is attributable to a rapid, temporary repression of transcription of the RP genes that apparently arises from a different cause than the repression connected with a failure of the secretory pathway (1).2 It is interesting that this heat shock effect is also independent of a functional protein kinase A pathway (Fig. 3, A and B, lane 6).

PKC1 Is Required for the Repression of RP Genes Caused by a Defect in the Secretory Pathway-- In what way would failure of the secretory pathway lead to the repression of ribosome synthesis? Many sec-type mutations in genes encoding components of the secretory pathway lead to almost immediate inhibition of secretion after the culture is shifted to the nonpermissive temperature (53). However, protein synthesis and growth, as estimated from turbidity measurements, continue for nearly a generation. The accumulation of intracellular proteins, under conditions in which there can be no new membrane synthesis, should lead to membrane stretch or osmotic stress, particularly in the rapidly growing bud. One way to mimic such a stretch effect is with CPZ, which inserts into the plasma membrane (54). CPZ was added to a culture of S. cerevisiae, and samples were harvested at increasing times to measure the levels of RNAs (Fig. 4). Comparison with the stable U3 snoRNA demonstrates that over 120 min there is little effect on the level of ACT1 mRNA, the intrinsic t1/2 of which is ~30 min (55).2 In contrast CPZ causes a rapid loss of RPL30 mRNA. Because the t1/2 of RPL30 mRNA is 7-10 min,2 the data of Fig. 4 demonstrate that the transcription of the RP gene is repressed almost immediately. This result suggests a tight connection between events at the plasma membrane and the transcription of RP genes.


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Fig. 4.   Membrane stretching produced by treatment with chlorpromazine represses RP gene transcription. Strain W303 was grown overnight in YPD at 30 °C and then treated with chlorpromazine to a final concentration of 250 µM. Total RNA was isolated and analyzed by Northern blot using radiolabeled probes for RPL30, ACT1, or U3 as described under "Materials and Methods." 5 µg of total RNA was loaded in each lane. The graph shows the ratio of RPL30 to ACT1 mRNA as a function of time.

Do signal transduction pathways responding to the osmotic condition of the cell have an influence on the repression of RP transcription in response to a defect in secretion? There are two such pathways, the high osmotic growth pathway, which responds to high osmotic pressure from the exterior, and the PKC-mitogen-activated protein kinase pathway, which responds to high osmotic pressure from the interior or to defects in the plasma membrane (for review, see Refs. 20 and 56). We first used congenic strains with a deletion of either HOG1 or PBS2, encoding members of the high osmotic growth protein kinase cascade pathway that regulates the transcription of a set of genes in response to an increase in extracellular solutes (57, 58). Deletion of either HOG1 or PBS2 had no effect on the repression of transcription of RP genes in response to a temperature-sensitive mutation in SLY1 (data not shown).

The PKC pathway is a more likely candidate because it responds to perturbations of the plasma membrane (19, 54). A complicating factor is that pkc1Delta strains are osmotically sensitive and must be grown with osmotic support, usually 1 M sorbitol (25). The presence of 1 M sorbitol leads to phenotypic suppression of many temperature-sensitive sec mutants, including sly1-1 and ypt6-1, presumably because of stabilization of the mutant proteins. However, we find that strains carrying sec1-1 remain temperature-sensitive in the presence of 1 M sorbitol. Therefore, we generated a set of congenic strains with all four combinations of SEC1/sec1-1 and PKC1/pkc1Delta genes (Table I, MN51-MN54).

                              
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Table I
Yeast strains used in this work

Strains MN51-MN54 were shifted from 23 to 37 °C, and their RNA was analyzed (Fig. 5). It is apparent that the deletion of the PKC1 gene spares the repression of the RP genes that occurs because of a defect in sec1 (Fig. 5, compare lanes 7 and 8 with lanes 15 and 16). This occurs both for RP genes that use Rap1p as transcriptional activator (RPL8 and RPL30) as well as for an RP gene that uses Abf1p as a transcriptional activator (RPS28). Note that because all the cultures shown in Fig. 5 were grown in the presence of 1 M sorbitol, the osmotic support itself cannot explain the loss of response to the mutant sec1-1 allele. However, it is also apparent from Fig. 5 that Pkc1p is not required for the loss of RP mRNA attributable to heat shock (Fig. 5, compare lanes 2 and 10). This result is further evidence that the repression of RP transcription attributable to a heat shock occurs by a different pathway than the one that responds to a secretion defect.


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Fig. 5.   pkc1Delta abrogates repression because of a secretion defect. The strains indicated below were grown overnight in YPD plus 1 M sorbitol at 23 °C and then shifted to 37 °C. Total RNA was isolated and analyzed by Northern blot using radiolabeled probes for ACT1, RPL3, RPL8, RPL30, RPS28, or U3. 5 µg of total RNA was loaded in each lane. The genotypes listed above the lanes represent the following strains (see Table I): SEC1 PKC1, MN51; sec1-1 PKC1, MN52; SEC1 pkc1Delta , MN53; and sec1-1 pkc1Delta , MN54.

The repression of ribosome synthesis in response to a defect in the secretory pathway is manifest not only in the transcription of the RP genes but also in the repression of transcription of rRNA (1). To ask whether Pkc1p is involved in repression of rRNA transcription, we used a pulse-chase of [C3H3]methionine, which can be rapidly converted to S-adenosyl methionine, which donates the C3H3 group to newly transcribed rRNA (59). Newly formed 35 S precursor RNA is rapidly methylated at numerous 3'-OH sites and is subsequently processed through 32-27 and 20 S intermediates, which are then processed to the 25 and 18 S mature RNA molecules. This is apparent in Fig. 6, lanes 1-3 and 7-9, which shows a 3-min pulse of [C3H3]methionine followed by a chase with cold methionine for 3 and 10 min. After the pulse, rRNA precursors and intermediates, but very little 25 and 18 S mature rRNA, are present (Fig. 6, lanes 1 and 7). During the chase, (Fig. 6, lanes 2 and 3 and 8 and 9) nearly all the radioactive rRNA is converted to the mature forms. By contrast, the sec1 strain, after 90 min at 37, incorporates <10% of the normal amount of [C3H3] into rRNA (Fig. 6., lane 4). The little rRNA that is transcribed is processed nearly as well as in wild-type cells (Fig. 6., lanes 5 and 6). However in a sec1-1 pkc1Delta strain, repression of rRNA transcription does not occur (Fig. 6., compare lanes 7 and 10); furthermore, processing of the precursor to the mature rRNA continues normally (Fig. 6., compare lanes 8 and 11).


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Fig. 6.   rRNA synthesis in sec1-1 PKC or sec1- pkc1Delta strains. Strains MN53 and MN54 were grown to early log phase (A600 = 0.75) in synthetic medium without Met containing 1 M sorbitol at 23 °C. Aliquots from each culture were shifted to 37 °C for 90 min. Each culture was pulse-labeled with [C3H3]methionine (60 µCi/ml) for 3 min, which was chased with nonradioactive methionine (500 µg/ml). Samples were chilled on crushed sterile ice at the time of addition of cold methionine (t = 0) and after chase times of 3 and 10 min. Total RNA was prepared, and 20 µg of each sample was analyzed by electrophoresis, followed by fluorography as described under "Materials and Methods."

These results demonstrate that deletion of PKC1 prevents the repression of the rRNA and RP genes, i.e. has a positive effect on transcription. This is strong evidence that the absence of Pkc1p does not simply add to the deterioration of the cell. Thus, Pkc1p appears to be an integral part of the pathway that connects a failure in secretion to the repression of ribosome synthesis, as manifest in the transcriptional activity not only of RP genes but of rRNA genes as well. This coordination of rRNA and RP transcription is characteristic of the regulation of ribosome biosynthesis (for review, see Refs. 3 and 9).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Defects in the yeast secretory pathway, from a step before insertion of peptide into the ER (sec63) to a step involving vesicle fusion with the plasma membrane (sec1), lead to the repression of ribosome synthesis (1). For this to occur, three types of events are necessary. Some element of the cell detects the defect in the secretory pathway. This detection leads to a signal transduction pathway. This pathway interacts with elements of the transcriptional apparatus.

Formally, one could argue that ribosome synthesis depends directly on the transit of an essential protein through the secretory pathway. If so, inhibition of protein synthesis should exacerbate the repression. In fact, the opposite effect was found (1). Because protein synthesis is necessary for the repression, we suggest that it is attributable, at least indirectly, to the continued accumulation of new protein in the absence of a functioning secretory pathway. The most likely scenario is that a defect in the secretory pathway rapidly leads to a defect in some component of the cell wall and/or the plasma membrane, both of which are composed of proteins that have passed through the entire pathway. Consequently, these would be vulnerable to a defect anywhere in the secretory pathway; indeed, a mutant Sec1p, responsible for the fusion of membrane vesicles with the plasma membrane (60), leads to repression of ribosome synthesis at the restrictive temperature (Fig. 3A and Ref. 1). On the other hand, secretory mutants of the vps class, such as pep12Delta , or a temperature-sensitive allele of vps18 (61), which participate in a branch of the secretory pathway that leads from the Golgi to the vacuole, have no effect on ribosome synthesis (data not shown).

In the experiments described above, we have shown that neither the regulatory systems involved in detecting problems in the ER nor common mechanisms known to regulate ribosome biosynthesis are involved in the interaction between the secretory pathway and ribosome synthesis. Rather, the effects of a failure in the secretory pathway appear to be detected at the plasma membrane. This is manifest in the hypersensitivity of transcription of RP genes to CPZ (Fig. 4), an agent that causes membrane stretch, and in the failure of cells lacking Pkc1p to repress rRNA and RP transcription in response to a secretory defect (Figs. 5 and 6).

In the cells of metazoans, a family of PKC isozymes can interact with specific downstream elements to effect a variety of responses (for review, see Refs. 62 and 63). In S. cerevisiae, however, only a single PKC pathway has so far been observed, passing through Bck1p, Mkk1,2p, and Mpk1p (for review, see Ref. 20). Nevertheless, this pathway not only monitors the integrity of the plasma membrane but is also involved in cell cycle regulation and bud emergence (64, 65). It has a number of inputs. Rho1p is a ras family GTPase that is implicated in bud growth (66) and in heat shock activation, acting through the Pkc1p pathway (67). Rho1p itself is downstream from the phosphatidytlinositol kinase homologue, Tor2p (68), which responds to nutrient availability and also helps organize the actin cytoskeleton (69). A group of membrane proteins, Wsc1-4p, have been implicated in transducing stress from the plasma membrane to the Pkc1p pathway (70). It remains to be seen which of these inputs senses the primary effect of a defect in the secretory pathway.

Indeed, the effects of a deletion of PKC1 are greater than that of its known downstream elements, leading to the suggestion that there is likely to be a bifurcation downstream of Pkc1p (71). Perhaps signaling to the ribosome biosynthetic apparatus is the responsibility of that other pathway. The control of ribosome biosynthesis during stress is not a peripheral consideration for the cell. Halting the synthesis of ribosomes in response to stress at the plasma membrane has two potentially life-saving effects: it frees a great deal of ATP, consumed in the synthesis of ribosomes, for use in metabolic reactions to protect the plasma membrane; and it reduces the exponential increase in protein production, thus slowing the accumulating intracellular pressure on the plasma membrane.

In retrospect, it is not surprising that the plasma membrane is an important regulatory element. As the major interface between intracellular and extracellular space, its maintenance is of critical importance. In a growing S. cerevisiae the biosynthetic capacity of the entire cell contributes to filling the bud, requiring a continuous, rapid expansion of its plasma membrane. We tested two situations in which there might be partial sparing of the need for new membrane in the bud. Endocytosis cycles plasma membrane material back into the cell, withdrawing it from the plasma membrane. Mutations in END3 or END4 reduce endocytosis (72). Such mutations might partially suppress the stress on the plasma membrane in a sly1-1 mutant at the nonpermissive temperature, thus reducing the repression of RP transcription. They did not (data not shown). Mutations in CDC42 interrupt normal bud formation, leading to spherical growth of the cell (73). We reasoned that this might partially spare the effect of a sly1-1 mutant, because the stretching of the plasma membrane would be distributed over the entire cell surface. It did not (data not shown). This result suggests that there is a continuum of the plasma membrane through the bud neck, such that an insufficiency of plasma membrane material is distributed over the entire cell surface.

Ultimately a signal transduction pathway must have an effect on transcription. Most RP genes are activated through two adjacent Rap1p binding sites (28, 41, 42). A few, such as RPL3 and RPS28, are activated by the binding of Abf1p (43, 74) Both classes are repressed by a failure of the secretory pathway (Ref. 1 and Figs. 1, 3, and 5). However, mutations of RAP1 that delete the silencing region block the repression of both classes of genes (75), suggesting that Rap1p can repress RP genes without binding to the DNA of the upstream activating region. This deduction is supported by our recent finding that sequences of RPL30 that lie between the Rap1p binding sites and the site of initiation of transcription make a substantial contribution to the repression that results from a defect in the secretory pathway.2 Conditions that produce changes in the levels of mRNAs encoding RPs do not significantly change the levels of expression of Rap1p (16), or change the binding affinities of Rap1p and Abf1p (12, 14, 16). Furthermore, cells that are defective in the silencing function of Rap1p respond normally to the effects of heat shock (75). This combination of results implies that the regulation works at the level of coactivator proteins that interact with Rap1p and/or Abf1p, rather than with the general activators themselves.

We conclude that the secretory pathway is connected to the repression of ribosome biosynthesis through a series of steps. A failure of the secretory pathway results in insufficient supply of material to continue growth of the plasma membrane. Because protein synthesis continues, an imbalance develops, causing intracellular pressure that leads to stretching of the plasma membrane. That pressure activates Pkc1p, which in turn activates a downstream pathway. A (the) target of this pathway is Rap1p, which represses, in some way that is not yet clear, the transcription of RP genes (75).2 Although there remain many gaps in our dissection of this pathway, it is experimentally supported and physiologically reasonable.

It is apparent from Fig. 3 that even in wild-type cells subjected to a mild heat shock, between two temperatures that support active growth, there is a striking, but temporary, reduction of mRNA encoding RPs. Analysis of mRNA levels by in vitro translation showed that such heat shock repression was true of nearly all of the >40 RPs examined (76). A recent genome-wide determination of mRNA levels extended this observation to nearly all of the 137 RP genes (77). Although this response of the RP genes to heat shock appears superficially similar to the response to a defect in the secretory pathway, they are the result of quite different pathways. The heat shock response is essentially immediate, whereas the response to the secretory pathway takes some time and appears to depend on protein synthesis (1). The response to the secretory pathway depends on the silencing region of Rap1p, whereas the heat shock response does not (75). Finally, repression of RP gene transcription in response to heat shock does not depend on Pkc1p (Fig. 5). This observation is intriguing because one of the roles of the PKC pathway in S. cerevisiae is to affect at least some aspects of the heat shock response (54). A major downstream target of the PKC pathway is the kinase Slt1p, the activity of which increases 100-fold in response to heat shock (54). However, this increase develops only after ~20 min, by which time the transcription of RP genes is returning to normal.

The importance of the transcription of the 137 RP genes to the economy of the cell is apparent from the existence of at least three distinct pathways that can effect the repression of this set of genes. The protein kinase A pathway represses the RP genes in response to carbon source (16, 18). A PKC pathway represses the RP genes in response to a defect in the secretory pathway, probably in its role of monitoring the integrity of the plasma membrane. An as yet unknown pathway represses the RP genes in response to heat shock.

Three intriguing questions remain. Is there a single molecular "switch" that is ultimately responsible for turning on or turning off the 50% of the mRNA transcription of the cell represented by the RP genes? Both bacterial (78) and metazoan (79) cells regulate their production of RPs primarily at the level of translation in very different ways. Why has S. cerevisiae evolved to do so at the level of transcription? What elements connect the regulation of transcription of RP genes by Pol II with the transcription of the rRNA genes by Pol I?

    ACKNOWLEDGEMENTS

We are grateful to R. Ballester, D. Levin, T. Michaeli, P. Novick, R. Sheckman and P. Walter for strains and plasmids and to D. Levin, T. Michaeli, K. Mizuta, and J. Vilardell for valuable discussions.

    Note Added in Proof

We have recently found that the repression of RP genes in response to tunicamycin is blocked in strains with a deletion of WSC1 (HCS77), which encodes a transmembrane protein thought to initiate the PKC pathway (Verna, J., Lodder, A., Lee, K., Vagts, A., and Ballester, R., (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13804-13809; Gray, J. V., Ogas, J. P., Kamada, Y., Stone, M., Levin, D. E., and Herskowitz, I. (1997) EMBO J. 16, 4924-4937). We suggest that Wsc1p is responsible for detecting the disturbance within the plasma membrane that occurs when the secretory pathway fails.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants GM25532 (to J. R. W.) and CA13330 (to the Albert Einstein Cancer Center).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present Address: Juvenile Diabetes Foundation International, 120 Wall St., New York, NY 10005.

§ To whom correspondence should be addressed: Dept. of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-3022; Fax: 718-430-8574; E-mail: warner{at}aecom.yu.edu.

2 B. Li, C. R. Nierras, and J. R. Warner, submitted for publication.

3 A new nomenclature for ribosomal proteins and their genes was recently adopted (http://speedy.mips.biochem.mpg.de/mips/yeast/) (7). For this paper, we have used the new names for ribosomal proteins throughout. Thus, L30 was previously known as L32; L3 was previously known as Tcm1p; L8 was previously known as protein L4; and S28 was previously known as S33.

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; RP, ribosomal protein; UPR, unfolded protein response; PKC, protein kinase C; CPZ, chlorpromazine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
  1. Mizuta, K., and Warner, J. R. (1994) Mol. Cell. Biol. 14, 2493-2502[Abstract]
  2. Li, B., and Warner, J. R. (1996) J. Biol. Chem. 271, 16813-16819[Abstract/Free Full Text]
  3. Woolford, J. L., Jr., and Warner, J. R. (1991) in The Molecular and Cellular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics (Broach, J. R., Pringle, J. R., and Jones, E. W., eds), pp. 587-626, Cold Spring Harbor Laboratory Press, New York
  4. Velculescu, V. E., Zhang, L., Zhou, W., Vogelstein, J., Basrai, M. A., Bassett, D. E., Jr., Hieter, P., Vogelstein, B., and Kinzler, K. W. (1997) Cell 88, 243-251[Medline] [Order article via Infotrieve]
  5. Holstege, F. C. P., Jennings, E. G., Wyrick, J. J., Lee, T. I., Hengartner, C. J., Green, M. R., Golub, T. R., Lander, E. S., and Young, R. A. (1998) Cell 95, 717-728[Medline] [Order article via Infotrieve]
  6. Thomas, B. J., and Rothstein, R. (1989) Cell 56, 619-630[Medline] [Order article via Infotrieve]
  7. Mager, W. H., Planta, R. J., Ballesta, J. P., Lee, J. C., Mizuta, K., Suzuki, K., Warner, J. R., and Woolford, J. L., Jr. (1997) Nucleic Acids Res. 25, 4872-4875[Abstract/Free Full Text]
  8. Warner, J. R. (1989) Microbiol. Rev. 53, 256-271
  9. Planta, R. J. (1997) Yeast 13, 1505-1518[CrossRef][Medline] [Order article via Infotrieve]
  10. Ju, Q., and Warner, J. R. (1994) Yeast 10, 151-157[Medline] [Order article via Infotrieve]
  11. Kief, D. R., and Warner, J. R. (1981) Mol. Cell. Biol. 1, 1007-1015[Medline] [Order article via Infotrieve]
  12. Kraakman, L. S., Griffioen, G., Zerp, S., Groeneveld, P., Thevelein, J. M., Mager, W. L., and Planta, R. J. (1993) Mol. Gen. Genet. 239, 196-204[Medline] [Order article via Infotrieve]
  13. Warner, J. R., and Gorenstein, C. (1978) Nature 275, 338-339[Medline] [Order article via Infotrieve]
  14. Moehle, C. M., and Hinnebusch, A. G. (1991) Mol. Cell. Biol. 11, 2723-2735[Medline] [Order article via Infotrieve]
  15. Broach, J. R. (1991) Trends Genet. 7, 28-33[CrossRef][Medline] [Order article via Infotrieve]
  16. Klein, C., and Struhl, K. (1994) Mol. Cell. Biol. 14, 1920-1928[Abstract]
  17. Shamu, C. E., Cox, J. S., and Walter, P. (1994) Trends Cell Biol. 4, 56-60[CrossRef]
  18. Neuman-Silberberg, F. S., Bhattacharya, S., and Broach, J. R. (1995) Mol. Cell. Biol. 15, 3187-3196[Abstract]
  19. Levin, D. E., and Errede, B. (1995) Curr. Opin. Cell Biol. 7, 197-202[CrossRef][Medline] [Order article via Infotrieve]
  20. Gustin, M. C., Albertyn, J., Alexander, M., and Davenport, K. (1998) Microbiol. Mol. Biol. Rev. 62, 1264-1300[Abstract/Free Full Text]
  21. Rose, M. D., Winston, F., and Hieter, P. (1990) Methods in Yeast Genetics. A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, New York
  22. Ito, H., Fukuda, K., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163-168[Medline] [Order article via Infotrieve]
  23. Nikawa, J.-I., and Yamashita, S. (1992) Mol. Microbiol. 6, 1441-1446[Medline] [Order article via Infotrieve]
  24. Cameron, S., Levin, L., Zoller, M., and Wigler, M. (1988) Cell 53, 555-566[Medline] [Order article via Infotrieve]
  25. Levin, D. E., and Bartlett-Heubusch, E. (1992) J. Cell Biol. 116, 1221-1229[Abstract]
  26. Rose, M. D., Novick, P., Thomas, J. H., Botstein, D., and Fink, G. R. (1987) Gene (Amst.) 60, 237-243[CrossRef][Medline] [Order article via Infotrieve]
  27. Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucleic Acids Res. 18, 3091-3092[Medline] [Order article via Infotrieve]
  28. Rotenberg, M. O., and Woolford, J. L., Jr. (1986) Mol. Cell. Biol. 6, 674-687[Medline] [Order article via Infotrieve]
  29. Udem, S. A., and Warner, J. R. (1972) J. Mol. Biol. 65, 227-242[Medline] [Order article via Infotrieve]
  30. Sidrauski, C., Chapman, R., and Walter, P. (1998) Trends Cell Biol. 8, 245-249[CrossRef][Medline] [Order article via Infotrieve]
  31. Shamu, C. E. (1998) Curr. Biol. 8, R121-R123[Medline] [Order article via Infotrieve]
  32. Cox, J. S., and Walter, P. (1996) Cell 87, 391-404[Medline] [Order article via Infotrieve]
  33. Sidrauski, C., and Walter, P. (1997) Cell 90, 1031-1039[Medline] [Order article via Infotrieve]
  34. Normington, K., Kohno, K., Kozutsumi, Y., Gething, M.-J., and Sambrook, J. (1989) Cell 57, 1223-1236[Medline] [Order article via Infotrieve]
  35. Rose, M. D., Misra, L. M., and Vogel, J. P. (1989) Cell 57, 1211-1221[Medline] [Order article via Infotrieve]
  36. Mori, K., Ogawa, N., Kawahara, T., Yanagi, H., and Yura, T. (1998) J Biol. Chem. 273, 9912-9920[Abstract/Free Full Text]
  37. Cox, J. S., Shamu, C. E., and Walter, P. (1993) Cell 73, 1197-1206[Medline] [Order article via Infotrieve]
  38. Mori, K., Ma, W., Gething, M. J., and Sambrook, J. (1993) Cell 74, 743-756[Medline] [Order article via Infotrieve]
  39. Dascher, C., Ossig, R., Gallwitz, D., and Schmitt, H. D. (1991) Mol. Cell. Biol. 11, 872-885[Medline] [Order article via Infotrieve]
  40. Ossig, R., Dascher, C., Trepte, H. H., Schmitt, H. D., and Gallwitz, D. (1991) Mol. Cell. Biol. 11, 2980-2993[Medline] [Order article via Infotrieve]
  41. Schwindinger, W. F., and Warner, J. R. (1987) J. Biol. Chem. 262, 5690-5695[Abstract/Free Full Text]
  42. Woudt, L. P., Smit, A. B., Mager, W. H., and Planta, R. J. (1986) EMBO J. 5, 1037-1040[Abstract]
  43. Hamil, K. G., Nam, H. G., and Fried, H. M. (1988) Mol. Cell. Biol. 8, 4328-4341[Medline] [Order article via Infotrieve]
  44. Jensen, K. F., and Pedersen, S. (1990) Microbiol. Rev. 54, 89-100
  45. Griffioen, G., Mager, W. H., and Planta, R. J. (1994) FEMS Microbiol. Lett. 123, 137-144[CrossRef][Medline] [Order article via Infotrieve]
  46. DeRisi, J. L., Iyer, V. R., and Brown, P. O. (1997) Science 278, 680-686[Abstract/Free Full Text]
  47. Thevelein, J., and Beullens, M. (1985) J. Gen. Microbiol. 131, 3199-3209[Medline] [Order article via Infotrieve]
  48. Thevelein, J. (1994) Yeast 10, 1753-1790[Medline] [Order article via Infotrieve]
  49. Toda, T., Uno, I., Ishikawa, T., Powers, S., Kataoka, T., Broek, D., Cameron, S., Broach, J., Matsumoto, K., and Wigler, M. (1985) Cell 40, 27-36[Medline] [Order article via Infotrieve]
  50. Kataoka, T., Powers, S., McGill, C., Fasano, O., Strathern, J., Broach, J., and Wigler, M. (1984) Cell 37, 437-445[Medline] [Order article via Infotrieve]
  51. Toda, T., Cameron, S., Sass, P., Zoller, M., Scott, J. D., McMullen, B., Hurwitz, M., Krebs, E. G., and Wigler, M. (1987) Mol. Cell. Biol. 7, 1371-1377[Medline] [Order article via Infotrieve]
  52. Kim, C. H., and Warner, J. R. (1983) Mol. Cell. Biol. 3, 457-465[Medline] [Order article via Infotrieve]
  53. Wooding, S., and Pelham, H. R. B. (1998) Mol. Biol. Cell 9, 2667-2680[Abstract/Free Full Text]
  54. Kamada, Y., Jung, U. S., Piotrowski, J., and Levin, D. E. (1995) Genes Dev. 9, 1559-1571[Abstract]
  55. Herrick, D., Parker, R., and Jacobson, A. (1990) Mol. Cell. Biol. 10, 2269-2284[Medline] [Order article via Infotrieve]
  56. Davenport, K. R., Sohaskey, M., Kamada, Y., and Levin, D. E. (1995) J Biol. Chem. 270, 30157-30161[Abstract/Free Full Text]
  57. Brewster, J. L., de Valoir, T., Dwyer, N. D., Winter, E., and Gustin, M. C. (1993) Science 259, 1760-1763[Medline] [Order article via Infotrieve]
  58. Brewster, J. L., and Gustin, M. C. (1994) Yeast 10, 425-439[Medline] [Order article via Infotrieve]
  59. Warner, J. R., Morgan, S. A., and Shulman, R. W. (1976) J. Bacteriol. 125, 887-891[Medline] [Order article via Infotrieve]
  60. Novick, P., Field, C., and Schekman, R. (1980) Cell 21, 205-215[Medline] [Order article via Infotrieve]
  61. Rieder, S. E., and Emr, S. D. (1997) Mol. Biol. Cell 8, 2307-2327[Abstract/Free Full Text]
  62. Mochly-Rosen, D., and Gordon, A. S. (1998) FASEB J. 12, 35-42[Abstract/Free Full Text]
  63. Ktistakis, N. T. (1998) Bioessays 20, 495-504[CrossRef][Medline] [Order article via Infotrieve]
  64. Madden, K., Sheu, Y.-J., Baetz, K., Andrews, B., and Snyder, M. (1997) Science 275, 1781-1784[Abstract/Free Full Text]
  65. Gray, J. V., Ogas, J. P., Kamada, Y., Stone, M., Levin, D. E., and Herskowitz, I. (1997) EMBO J. 16, 4924-4937[Abstract/Free Full Text]
  66. Yamochi, W., Tanaka, K., Nonaka, H., Maeda, A., Musha, T., and Takai, Y. (1994) J. Cell Biol. 125, 1077-1093[Abstract]
  67. Kamada, Y., Qadota, H., Python, C. P., Anraku, Y., Ohya, Y., and Levin, D. E. (1996) J Biol. Chem. 271, 9193-9196[Abstract/Free Full Text]
  68. Schmidt, A., Bickle, M., Beck, T., and Hall, M. N. (1997) Cell 88, 531-542[Medline] [Order article via Infotrieve]
  69. Helliwell, S. B., Howald, I., Barbet, N., and Hall, M. N. (1998) Genetics 148, 99-112[Abstract/Free Full Text]
  70. Verna, J., Lodder, A., Lee, K., Vagts, A., and Ballester, R. (1997) Proc. Natl. Acad. Sci. USA 94, 13804-13809[Abstract/Free Full Text]
  71. Errede, B., and Levin, D. E. (1993) Curr. Opin. Cell Biol. 5, 254-260[Medline] [Order article via Infotrieve]
  72. Raths, S., Rohrer, J., Crausaz, F., and Riezman, H. (1993) J. Cell Biol. 120, 55-65[Abstract]
  73. Adams, A. E. M., Johnson, D. I., Longnecker, R. M., Sloat, B. F., and Pringle, J. R. (1990) J. Cell Biol. 111, 131-142[Abstract]
  74. Herruer, M. H., Mager, W. H., Doorenbosch, T. M., Wessels, P. L., Wassenaar, T. M., and Planta, R. J. (1989) Nucleic Acids Res. 17, 7427-7439[Abstract]
  75. Mizuta, K., Tsujii, R., Warner, J. R., and Nishiyama, M. (1998) Nucleic Acids Res. 26, 1063-1069[Abstract/Free Full Text]
  76. Warner, J. R., and Gorenstein, C. (1977) Cell 11, 201-212[Medline] [Order article via Infotrieve]
  77. Eisen, M. B., Spellman, P. T., Brown, P. O., and Botstein, D. (1998) Proc. Natl. Acad. Sci. USA 95, 14863-14868[Abstract/Free Full Text]
  78. Zengel, J. M., and Lindahl, L. (1994) Prog. Nucleic Acid Res. Mol. Biol. 47, 331-370[Medline] [Order article via Infotrieve]
  79. Meyuhas, O., Avni, D., and Shama, S. (1996) in Translational Control (Hershey, J. W. B., Mathews, M. B., and Sonenberg, N., eds), pp. 363-388, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY


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