From the Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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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.
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.
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 ire1
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 pkc1 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
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 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
To visualize the C3H3-labeled RNA, agarose gels
were soaked in En3Hance (DuPont NEN), washed in water,
dried, and visualized by radiography.
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/ire1
As an alternative, the wild-type strain carrying ire1 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.
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).
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.
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 pkc1
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.
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 pkc1
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).
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 pep12 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?
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
::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 ire1
::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.
, and the deletion was confirmed by testing for the requirement of pkc1
strains for osmotic support
(1 M sorbitol) (25).
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.
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.
70 °C. RNA was prepared and analyzed on agarose
gels as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
SLY1/sly1-1 diploid led to no viable
ire1
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.
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
ire1 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.
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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.
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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 bcy1 ,
MN16; sly1-1 BCY1, MN14; and sly1-1 bcy1
,
MN13.
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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.
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/pkc1
genes (Table
I, MN51-MN54).
Yeast strains used in this work
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Fig. 5.
pkc1 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 pkc1
, MN53; and sec1-1 pkc1
,
MN54.
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- pkc1 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."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, 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).
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ACKNOWLEDGEMENTS |
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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.
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Note Added in Proof |
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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, 1380413809; 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.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; RP, ribosomal protein; UPR, unfolded protein response; PKC, protein kinase C; CPZ, chlorpromazine.
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REFERENCES |
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