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J. Biol. Chem., Vol. 277, Issue 40, 37559-37566, October 4, 2002
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From the Department of Molecular Sciences, University of Tennessee,
Memphis, Tennessee 38163
Received for publication, May 17, 2002, and in revised form, June 26, 2002
Regulated intracellular localization of Gln3, the
transcriptional activator responsible for nitrogen catabolite
repression (NCR)-sensitive transcription, permits Saccharomyces
cerevisiae to utilize good nitrogen sources (e.g.
glutamine and ammonia) in preference to poor ones
(e.g. proline). During nitrogen starvation or growth in
medium containing a poor nitrogen source, Gln3 is nuclear and
NCR-sensitive transcription is high. However, when cells are grown in
excess nitrogen, Gln3 is localized to the cytoplasm with a concomitant
decrease in gene expression. Treating cells with the Tor protein
inhibitor, rapamycin, mimics nitrogen starvation. Recently, carbon
starvation has been reported to cause nuclear localization of Gln3 and
increased NCR-sensitive transcription. Here we show that nuclear
localization of Gln3 during carbon starvation derives from its indirect
effects on nitrogen metabolism, i.e. Gln3 does not move
into the nucleus of carbon-starved cells if glutamine rather than
ammonia is provided as the nitrogen source. In addition, these studies
have clearly shown Gln3 is not uniformly distributed in the cytoplasm,
but rather localizes to punctate or tubular structures. Analysis of
these images by deconvolution microscopy suggests that Gln3 is
concentrated in or associated with a highly structured system in the
cytosol, one that is possibly vesicular in nature. This finding may
impact significantly on how we view (i) the mechanism by which Tor
regulates the intracellular localization of Gln3 and (ii) how proteins
move into and out of the nucleus.
The budding yeast Saccharomyces cerevisiae is often
used as a model with which to elucidate the functions of important
mammalian proteins. Few such proteins have generated greater excitement than Tor1/2 and their mammalian counterpart mTor, which are inhibited by the immunosuppressant and antineoplastic drug rapamycin (1-5). Tor1
and the essential Tor2 proteins are reported to be situated at the top
of the Tor signal transduction cascade and through it to regulate an
exceedingly large number of diverse cellular processes, including:
translational initiation, G1-phase progression, autophagy,
RNA polymerase I/III function, actin cytoskeleton organization, membrane protein stability, nitrogen catabolite repression
(NCR)1-sensitive and
retrograde gene expression (1-9). In this work, we focus on the
last two of these processes.
NCR is the physiological process by which S. cerevisiae
selectively uses good nitrogen sources in its environment in preference to poor ones (5, 10-12). In the presence of good nitrogen sources (glutamine, asparagine, and in some strains, ammonia), expression of
genes encoding the permeases and enzymes needed for utilization of poor
nitrogen sources is held at low levels, i.e. expression is
repressed. When only poor (proline) or limiting amounts of good
nitrogen sources are available, these genes are then expressed at the
higher levels needed to scavenge whatever is available in the
environment (5, 10-12). Two GATA family transcriptional activators
(Gln3 and Gat1/Nil1) are responsible for NCR-sensitive transcription
and Ure2 (a yeast prion precursor) inhibits their ability to carry out
this function (5, 10-12). In glucose-proline medium, Gln3 and Gat1 are
bound to the GATA sequences of the NCR-sensitive CAN1 gene,
whereas in glucose-glutamine medium or in a
gln3 Work in four laboratories, using the immunosuppressant drug, rapamycin,
has provided insights into the mechanism regulating intracellular
localization of Gln3 (14-17). They found that inhibiting Tor1/2 with
rapamycin resulted in dephosphorylation of Gln3 and, in some
laboratories, Ure2, and its entry into the nucleus (14, 16, 17). Loss
of NCR-sensitive gene expression in rna1 and srp1
mutants (19, 20), led Carvalho et al. (20) to conclude that
these proteins are required for Gln3 entry into the nucleus and that
Crm1 mediates its exit. Tap42, a kinase that itself is phosphorylated
by Tor (21, 22), Tap41, type 2 phosphatases Sit4 and Pph3, Mks1 and
Ure2 are the remaining members of the Tor signal transduction cascade
reported to be situated between Tor and Gln3/Gat1 (1-3, 6-9, 14-18,
23, 24). Recent work (24), however, demonstrated that Mks1 functions
only indirectly in the regulation of Gln3/Gat1.
A second set of metabolic genes, those associated with the retrograde
response have also been reported to be regulated by the Tor signal
transduction cascade (6-9, 24-26). The retrograde genes are those
whose expression is increased in cells with damaged mitochondria
(27-30). These genes, including CIT2, ACO1,
IDH1/2, and DLD3, are thought to be responsible
for synthesizing The retrograde gene expression experiments just described collectively
demonstrate a relationship between nitrogen and carbon metabolism in
S. cerevisiae. Bertram et al. (31) have recently reported a second major bridge linking carbon and nitrogen metabolism. They reported that starving cells for carbon, like starving them for
nitrogen, results in Gln3 being localized to the nucleus and mediating
transcription of the NCR-sensitive genes, GAP1,
GDH2, and PUT1 (31). Carbon-starved nuclear
localization of Gln3 also correlates with its hyperphosphorylation.
Finally, Snf1 has been shown to be required for these carbon
starvation-induced changes in Gln3 localization, in phosphorylation,
and for the transcription it mediates (31).
Our interest was piqued by these carbon starvation experiments, because
here Gln3 is nuclear during the nitrogen excess, which exists during
carbon starvation. Investigating this phenomenon, we find that whether
or not Gln3 is localized to the nucleus during carbon starvation is
dictated by the nature of the nitrogen source, i.e. Gln3
nuclear localization occurs with ammonia but not glutamine as nitrogen
source. The effects of carbon starvation with ammonia as the nitrogen
source are indirect and most likely caused by the inability of ammonia
to be assimilated into glutamate during carbon starvation due to the
lack of Strains and Culture Conditions--
Strains used in this work
were JK9-3da (MATa, leu2-3,112,
ura3-52, rme1, trp1, his4,
GAL+, HMLa) and TB123
(MATa, leu2-3,112,
ura3-52, rme1, trp1, his4, GAL+, HMLa,
GLN3-myc[kanMX4]). Cultures were grown overnight at
30 °C in Difco YNB (without ammonium sulfate or amino acids), 2% glucose, and the indicated nitrogen source. Appropriate auxotropic requirements were supplied when media other than synthetic complete (SC) (31) were used. Experiments in which yeast were transferred from
one medium to another were performed as follows. A sample of the
exponentially growing cells (A600 nm = 0.4-0.7) was harvested from the initial medium for immunofluorescence
or RNA analysis. The remaining cells in the culture were collected, washed with the new medium, recollected, and resuspended in an equal
volume of the new medium. Additional samples were then collected and
processed for RNA isolation or immunofluorescence at the indicated times. Precisely the same format was also used for the transfer of
cells from rapamycin-free (initial medium) to rapamycin-containing (new
medium) media. Rapamycin (Sigma), from a concentrated stock solution in
90% ethanol/10% Tween 20, was used at a final concentration of 200 ng/ml where indicated.
RNA Isolation and Northern Blot Analysis--
Total RNA was
prepared from cultures of strain JK9-3da as previously described (24).
Total yeast RNA (9 µg) was separated on denaturing gels, transferred
to a nylon membrane, and hybridized with 32P-labeled DNA
probes, and the blots were washed as previously described (24).
Indirect Immunofluorescence--
Immunofluorescent staining of
yeast was carried out using a modification of the methods of Schwartz
et al. (32). Cultures of strain TB123 were fixed by the
addition of 1/10 volume of 37% formaldehyde and incubated with shaking
at 30 °C for 10 min. They were then collected by centrifugation at
room temperature and incubated in 3.7% formaldehyde in potassium
phosphate buffer (40 mM, pH 6.5, containing 0.5 mM MgCl2) for 1 h. After washing and Zymolyase digestion as previously described, cells were applied to
poly-L-lysine-coated microscope slides. The slides were
blocked overnight at 4 °C using 0.5% bovine serum albumin, 0.5%
Tween 20 in phosphate-buffered saline (pH 7.4). All further antibody incubations and washes were performed in this buffer. 9E10(c-myc) (Covance MMS-150P) was used at a dilution of 1:1000 as the primary antibody and either Texas Red-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) or Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes) was used at a dilution of 1:200 as the secondary antibody. To
visualize nuclei, 4',6'-diamino-2-phenylindole (DAPI) was added to the
mounting media at a final concentration of 50 µg/ml, and images were
collected immediately after mounting.
Immunofluorescence Microscopy--
Cells in Figs. 1 and 2 were
imaged using a Zeiss Axiophot microscope with a 63× Plan-Apochromat
1.40 oil objective. Images were acquired using AxioVision 3.0 (Zeiss)
software and a Zeiss Axio camera. Alternatively, cells shown in Figs.
7-9 (see below) were imaged using a Zeiss Axioplan 2 imaging
microscope with a 100× Plan-Apochromat 1.40 oil objective. Images were
acquired using a Zeiss Axio camera and deconvolved using AxioVision 3.0 (Zeiss) software using the constrained iterative algorithm.
Three-dimensional views (see Figs. 8, E-G, and 9 below)
were produced from 15 images in a two-dimensional Z-stack, spanning
about 1.5 µm of the center of a yeast cell using AxioVision Inside 4D
(Zeiss) software. Images were rendered using either the maximum
projection in which only pixels of the highest intensity along the axis
are displayed (Fig. 8, E-G) or the surface mode in which
non-transparent surfaces are calculated from gray values (Fig. 9).
Nitrogen Source Determines Intracellular Localization of Gln3
during Carbon Starvation--
A recent report indicates that carbon
starvation results in Snf1-dependent nuclear localization
of Gln3 (31). These data, coupled with earlier reports that nitrogen
starvation or limitation also results in nuclear localization of Gln3
and Gat1p (13-17), create a paradox. Cells grown in medium devoid of
nitrogen are by definition growing in carbon excess. Conversely, cells
grown in medium devoid of carbon are by definition growing in nitrogen excess. It was difficult to understand why Gln3 would react
similarly to physiologically opposite conditions, i.e.
nitrogen starvation and excess. Therefore, we investigated the
phenomenon in greater detail, first by performing and then extending
the critical published experiment (31). To acquire a crude estimate of
the time course of Gln3 redistribution, following perturbation, we
scored the intracellular localization of Gln3 in ~100 cells from
random fields collected at increasing times following the onset of
starvation. Multiple samples were independently scored in duplicate by
two different individuals. Insignificant variations were observed in
these duplicate counts. Following transfer of cells from SC to
glucose-free SC medium, little change was observed at 30 min (Fig.
1B, green bars);
representative microscopic images are shown in Fig. 1A.
Nuclear-localized Gln3 rose from 7% of the cells at 1 h to over
40% at 2 h, and up to about 60% at 3 h (Fig.
1B). These results are similar to the published work, except
that Gln3 response appeared to be slower in the strain we used. Gln3
appeared nuclear in the 30-min image of Bertram et al.
(31).
A principal consequence of carbon starvation is the loss of carbon
skeletons needed for biosynthetic reactions, such as those involving
glutamate and glutamine. The loss of the ability to synthesize these
amino acids would in turn generate serious consequences on nitrogen
metabolism and NCR. Therefore, we repeated the carbon starvation
experiment but accounted for this secondary consequence by performing
it in SC + 0.1% glutamine rather than SC medium. Even though the SC + glutamine medium, like that used above, was devoid of glucose, Gln3 was
nuclear in less than 10% of the cells at 2 h and decreased to
near undetectable levels thereafter (Fig. 1B, red
bars).
To ensure that the complex nitrogen mixture contained in SC medium was
not misleading us, we performed the experiment in YNB to which
was added a single nitrogen source, ammonia or glutamine. In
these less complex media, the time course profiles were even clearer.
Gln3 localized to the nuclei of a gradually increasing number of cells
cultured in carbon-free ammonia medium (Fig.
2B, green bars),
whereas with carbon-free glutamine medium, Gln3 remained cytoplasmic
throughout starvation. However, in contrast to SC medium in which
1 h was required for Gln3 to become nuclear in 7% of the cells,
in YNB-ammonia medium Gln3 was nuclear in 5% of the cells at the
outset. By 1 h, this value increased to 45% and reached
over 80% by the end of the experiment (Fig. 2B, green bars). With glutamine as sole nitrogen source, we were unable to
detect nuclear localization of Gln3 (Fig. 2B, red
bars). The data collectively argue that nuclear localization of
Gln3 is a function not of carbon starvation but of the nitrogen source
provided in the medium in which starvation is carried out. Carbon
starvation is inconsequential to the cellular localization of Gln3 if
sufficient glutamine is present, i.e. NCR is operating.
The Expression of Only Some NCR-sensitive Genes Correlates with the
Nuclear Localization of Gln3--
To determine the strength of
correlation between the intracellular Gln3 localization and
NCR-sensitive gene expression, we measured mRNA levels for several
well-studied nitrogen-related genes. Rather than use all of the
conditions we studied microscopically, the three most likely to be
informative were chosen: YNB-ammonia, YNB-glutamine, and SC media, with
samples harvested just prior to and 10, 30, and 120 min following the
onset of carbon starvation. The first thing we noticed was that genes
routinely used to assess loading and RNA transfer were not useful when
performing carbon starvation experiments. H3,
ACT1, and TCM1 mRNA levels all decreased following the onset of carbon starvation regardless of the medium used,
arguing that carbon starvation rather than nitrogen source was
responsible for the effects (Fig. 3,
B and C). The only probe we found usable as a
standard was one (designated pC4) used by investigators for studying
the onset of meiosis and sporulation. This standard also followed bulk
RNA in its quantitation.
Next we analyzed two highly NCR-sensitive genes, DAL5 and
DAL80. In both cases, expression reached detectable levels
only after 2 h of carbon starvation with ammonia as the nitrogen
source. DAL5 or DAL80 expression was not detected
in SC or YNB-glutamine grown cells (Fig. 3, A and
B). The GDH2 expression profile was quite
different. In YNB-ammonia medium GDH2 expression was low at
the outset of starvation, increased at 10 min, fell at 30 min, and
increased again at 2 h (Fig.
4A). In SC medium
GDH2 expression was detectable only after 2 h of carbon
starvation. Still different were the results with YNB-glutamine. Here,
GDH2 expression began low, increased at 10 min, decreased
marginally if at all at 30 min, and increased to very high levels after
2 h of starvation. On the other hand, expression of
NRG1, encoding a carbon-regulated gene, was high at the
outset of starvation and for the most part decreased thereafter
regardless of the nitrogen source employed (Fig. 4B).
The most striking characteristic of these data is the lack of a
consistent pattern of expression. At a more discriminating level,
however, there are visible patterns. If one considers a gene, known to
be regulated by carbon, NRG1, the pattern is reasonably consistent and largely independent of the nitrogen source. Similarly, with the gene whose expression has to date been demonstrated to be
regulated by nothing other than NCR (DAL5 or
DAL80), the pattern is again consistent and behaves as
predicted from the level of NCR generated by each nitrogen source. The
difficulty arises with genes like GDH2, assayed both in our
work and that of Betram et al. (31). GDH2
expression fluctuates in both laboratories, but not congruently.
However, when one takes into consideration that GDH2
expression is reported to be regulated in four distinctly different
ways, the variation is perhaps not so difficult to envision. As carbon
starvation progresses, intracellular levels of carbon and nitrogen
compounds are constantly changing. Because the level of GDH2
expression is the sum of four different types of regulation, such
fluctuation is expected rather than surprising. Therefore, unless the
gene chosen is subject to a single type of regulation whose response to
all of the experimental perturbations can be reliably predicted, data
like that seen with GDH2 are to be expected. Instances of
complex profiles can be traced to equally complex and differing modes
of regulation to which many nitrogen related genes are subjected, in
addition to NCR. For example, the CAR1 promoter has 14 distinct
promoter elements and 9 trans-acting factors that function during
expression (25).
Time Course of Gln3 Nuclear Import and Export--
Studies of the
mechanism by which NCR is accomplished have depended heavily upon the
ability of rapamycin-treatment to mimic nitrogen starvation (14-17).
The correlation of observations generated by nitrogen starvation and
rapamycin-treatment are indeed among the most important reasons for
concluding that Gln3 intracellular localization, and thereby NCR, are
regulated by the Tor1/2 signal cascade (6, 14-17). This prompted us to
compare the time course of events triggered by metabolic and
rapamycin-mediated perturbation. We first compared Gln3 distribution
following the onset of carbon starvation or after transfer of cells
from glucose-glutamine to glucose-proline media. Ammonia was used as a
nitrogen source for the carbon starvation experiment, because, with
glutamine, Gln3 isn't detected in the nucleus. As shown in Fig.
5, the two perturbations generate
time-course profiles that differ in two important respects. Gln3
movement into the nucleus becomes detectable between 3 and 6 min after
transferring glutamine-grown cells to proline and localizes there in
nearly 90% of the cells by 30 min (Fig. 5, green bars). In
glucose starvation the onset of Gln3 movement is slower, being first
detected at 30 min, and is far less complete, attaining localization in
about half of the cells at 120 min (Fig. 5). We suggest the different
profiles derive from the differences in the times required for the
metabolite that controls NCR to decrease sufficiently for NCR to be
relieved thereby permitting Gln3 to enter the nucleus. That more time
is required for glucose-generated
Equally pertinent comparisons are those made with rapamycin. Both
rapamycin and glutamine or the physiologically significant signal are
situated upstream of Tor1/2 in the signal transduction cascade
hypothesized to regulate the cellular distribution of Gln3 (6, 14-17).
Therefore, rapamycin should affect Gln3 distribution at least as
quickly or perhaps more so than a shift from glutamine to proline.
Three conditions were assayed to test this expectation: rapamycin
addition to cells provided with (i) ammonia or (ii) glutamine as
nitrogen source and (iii) transfer of cells from glutamine to proline.
Contrary to expectation, marked differences were again observed (Fig.
6). Gln3 becomes nuclear more quickly when cells are shifted from glutamine to proline (50% at 6 min) than
with rapamycin treatment irrespective of the nitrogen source (6% at 6 min with ammonia and 20% at 15 min with glutamine) (Fig. 6). Two
things are exceptional in these data: (i) the fact that rapamycin acts
more slowly than shifting glutamine-grown cells to proline, and (ii)
the speed with which rapamycin acts is dependent upon the nitrogen
source. The latter observation is also consistent with the fact that we
find rapamycin "induces" retrograde and NCR-sensitive gene
expression less well with glutamine than ammonia as the nitrogen source
(24). This would not be expected a priori, because rapamycin
is thought to act downstream of the signal metabolite.
The other striking difference is that transfer from glutamine to
proline medium generates a more sustained response than rapamycin, with
duration of the rapamycin effect again depending on the nitrogen source
used. Two hours after shifting cells from glutamine to proline, Gln3
was still localized to the nucleus in over 50% of the cells (Fig. 6,
green bars). In contrast, nuclear localization of Gln3
peaked at 15 and 30 min with ammonia and glutamine, respectively (Fig.
6, blue and red bars). Furthermore, Gln3
localization to the nuclei of rapamycin-treated, ammonia-grown cells
peaks earlier, is higher, and is sustained longer than when glutamine
is used (Fig. 6). With rapamycin-treatment, as with carbon starvation, the nitrogen source makes a noticeable difference in the time course of
Gln3 entry and exit from the nucleus even though rapamycin is
supposedly downstream of the sensed physiological signal.
Cytoplasmic Compartmentation of Gln3 during Nitrogen Catabolite
Repression--
During this work, we noticed that, when Gln3 was
localized to the cytoplasm, more often than not, the distribution of
fluorescence was not uniform. Gln3-derived fluorescence appeared to be
concentrated in "clumps" or more defined punctate structures;
several examples of this are shown in Fig.
7, row 3, images
A, C, E, and G). This "clumping" was very reminiscent of the bright, punctate foci
situated around the periphery of the cell or vacuole that we reported
earlier to occur with both EGFP-Gln3 and EGFP-Ure2 (see Figs. 9 and 10 of Ref. 13).
It was conceivable the punctate appearance observed in those earlier
studies derived from prion formation, because Ure2 was highly
overproduced (13). In contrast, the images in Figs. 1, 2, and 7 derive
from cells in which neither Ure2p nor Gln3 are overproduced, and so it
is unlikely that punctate staining derives from prion formation. These
data raised the possibility that Gln3 might not be uniformly
distributed throughout the cytosol during growth in good nitrogen
sources. To examine this more carefully, cultures were grown to mid-log
phase in either 2% glucose YNB medium containing 0.1% proline or
glutamine as nitrogen source. Cells in the proline-grown culture were
then transferred to medium containing glutamine as the nitrogen source.
At various times after the transfer, samples of the culture were taken
and analyzed by immunofluorescence microscopy. The glucose-glutamine
culture was not treated further before cell samples were taken.
Fluorescence is sometimes distributed as dots around the
periphery of the cell or ringing the vacuole (Fig. 7,
row 2, images B, D, F, and
H; row 3, images B, F, and
H) and at others as tubes or networks of tubes (row
2, images B, D, and F; row
3, images D, F, and H). As
expected, cells grown in glucose-proline medium exhibited nuclear localization of Gln3 (row 1). However, after only 1 min of
transfer to glucose-glutamine medium, about 50% of the cells showed
complete cytoplasmic localization of Gln3. In those cells that still
retained some nuclear concentration of Gln3, fluorescence can be seen
in projections emanating from the nucleus (row 2,
images B, D, and H). Note that the
images in Fig. 7 all appear as pairs, with fluorescent images (panels A, C, E, and
G) followed by deconvolved views (panels B,
D, F, and H).
To ascertain whether the fluorescent dots represented end views of
tubular structures, we analyzed three-dimensional reconstructions of
multiple images; an example is shown in Fig.
8. In the normal fluorescent image,
clearly defined dots appear to circle the cell (Fig. 8,
image A). They are much more apparent in the deconvolved reconstruction (image B, arrows). The nucleus was
visualized with DAPI staining (image C) and appears along
with Gln3 fluorescence in image D. As the plane of view is
rotated about its axes, fluorescent dots (arrows
in image B) turn into elongated tubes (Fig. 8,
images E-G, arrows).
Finally, the three-dimensional image from Fig. 8 was rendered using a
surface mode in which solid surfaces are constructed from the gray
values of the pixels (Fig. 9). In this
view, the structures appear similar to what would be expected if Gln3
was contained within or was attached to one of the vesicular systems of
the cell cytoplasm. The nucleus (blue image, stained with
DAPI) appears to come in close contact with several of the protrusions from this system. Although identity of the globular/tubular system responsible for the observed Gln3 distribution in the cytoplasm is not
yet clear, the images are consistent with the suggestion that a
significant fraction of Gln3 may not be not free in the cytoplasm but
rather localizes to a subcellular network during NCR.
We initiated this work to resolve an apparent paradox,
i.e. Gln3 is localized to the nucleus in both carbon and
nitrogen starvation (31). During carbon starvation, nitrogen is in
excess. That Gln3 is nuclear under conditions of nitrogen excess
prompted us to ask why Gln3 was cytoplasmic under some conditions of
nitrogen excess but not others.
We demonstrate that it's not carbon starvation per se that
results in Gln3 being localized to the nucleus but the indirect effects
of carbon starvation on nitrogen metabolism and hence on NCR. The
principal finding supporting this conclusion is that nuclear
localization of Gln3 observed upon transferring cells from SC to
carbon-free SC medium can be completely prevented if glutamine is added
to the latter medium. This result was confirmed using more defined
media containing single sources of nitrogen.
The time course of Gln3 localization points to a possible explanation
of the paradox. Gln3 moves more quickly to the nucleus and in a greater
percentage of the cells when cells are transferred from glutamine to
proline compared with transfer from glucose-ammonia to
glucose-free-ammonia medium. For ammonia to be assimilated into
nitrogenous compounds and thereby exert NCR on GATA-mediated transcription, it must first be converted to glutamate and glutamine. The carbon skeletons for these amino acids derive from glucose. As
glucose starvation worsens, carbon-containing compounds (including This is not the first instance in which nutrient starvation or
rapamycin-induced alteration of metabolite pools has generated indirect
effects on nitrogen catabolic gene expression. Tate et al.
(24) recently showed that Mks1, rather than being an important component of the Tor1/2 signal transduction pathway that positively regulates nuclear localization of Gln3 and Rtg1/3, is a negative regulator of Rtg1/3 function that only indirectly influences
NCR-sensitive transcription.
The time course of Gln3 movement from cytoplasm to nucleus is also
revealing from the standpoint of rapamycin-"induced"
GATA-factor-mediated gene expression. It is currently accepted that
Tor1/2, whose functions are inhibited by rapamycin, are situated
downstream of the metabolite effector of NCR in the nutrient limitation
signal transduction cascade (1-6, 14-17). Yet Gln3 movement into the
nucleus following transfer of cells from glutamine to proline medium is
faster, more complete, and longer lasting than in cells treated with
rapamycin. Furthermore, the time course profiles seen with ammonia and
glutamine following rapamycin addition are not the same. With
glutamine, Gln3 movement into the nucleus occurs more slowly, in fewer
cells, and remains nuclear for a shorter time than with ammonia.
In other words, the effect of rapamycin on Gln3 localization, as with
carbon starvation, depends on the nitrogen source. If Tor1/2 are indeed situated downstream of the nitrogen signal, the onset of Gln3 movement
into the nucleus should occur equally or more quickly with rapamycin
treatment and be independent of the nitrogen source. An important
caveat, we can't rigorously evaluate for lack of radioactive
rapamycin, is the possibility that rapamycin uptake is strongly
influenced by the nitrogen source. This, however, is unlikely in view
of the speed with which rapamycin is reported to work and the fact that
both ammonia and glutamine are good nitrogen sources in the strains we
used (14). Therefore, incongruities exist between expected and observed
time courses of rapamycin response when predictions are predicated on
current models of Tor1/2 signal transduction.
The finding that Gln3 localization may not be uniformly distributed in
the cytoplasm suggests an alternative view of the Tor1/2 signal
transduction cascade. We suggest that movement of Gln3 and Gat1 into
and out of the nucleus, like that of Rtg3, another phosphorylated
transcription factor reported to be regulated by the Tor1/2 signal
transduction pathway, involves the participation of a general protein
trafficking pathway. By our hypothesis, Tor1/2 may not be situated at
the top of the signal transduction pathway regulating NCR-sensitive and
retrograde gene expression but are participants in or regulators of the
general protein trafficking pathway through which Gln3/Gat1, Rtg1/3,
and a growing list of other unrelated transcription factors enter and
exit the nucleus. Just as Gln3 uses a generic nuclear import (Srp1) and
export (Crm1) proteins, which are also used by other unrelated
transcription factors, so too Gln3 may use a general protein
trafficking system whose components or regulators include Tor1/2. We
suggest the non-uniform distribution of Gln3 in the cytoplasm of
glutamine-grown cells may derive from its sequestration in or
localization to components of that general pathway.
Our challenge now is to determine whether the explanations suggested
above will stand the test of experimental investigation, and if they
do, to determine how they account for the large number of cellular
processes that are influenced, directly or indirectly, by the Tor1/2
proteins and their inhibition by rapamycin. Furthermore, if verified
experimentally, this interpretation of our data also provides a novel
way of viewing the nucleocytoplasmic shuttling of proteins into and out
of the nucleus.
We thank Dr. Michael Hall for very generously
providing strains, Dr. Ted Powers for sharing a preprint of his work,
Tim Higgins for preparing the artwork, and the University of Tennessee
Yeast Group for suggestions to improve the manuscript.
*
This work was supported by National Institutes of Health
Grant GM-35642.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.
Published, JBC Papers in Press, July 24, 2002, DOI 10.1074/jbc.M204879200
The abbreviations used are:
NCR, nitrogen
catabolite repression;
DAPI, 4',6-diamidino-2-phenylindole.
Cytoplasmic Compartmentation of Gln3 during Nitrogen Catabolite
Repression and the Mechanism of Its Nuclear Localization during Carbon
Starvation in Saccharomyces cerevisiae*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
gat1 strain, these GATAs are unoccupied
and available to serve as surrogate TATA elements (13). Correlating
with these observations, Gln3 and Gat1 are localized to the nucleus in
the former medium and to the cytoplasm in the latter (13).
-ketoglutarate, which is needed for glutamate and
its biosynthetic products when: (i) the normal tricarboxylic acid cycle
is shut down, (ii) cells are fermenting high concentrations of glucose,
or (iii) mitochondria are otherwise unable to function. Consistent with
this physiological function, retrograde gene expression is low when
cells are provided with glutamate as nitrogen source. Retrograde gene
expression is mediated by the transcription activators Rtg1/3 (24-30),
whose differential phosphorylation levels and intracellular
localization are regulated in a remarkably analogous way to those of
Gln3 and Gat1. Like Gln3 and Gat1, nuclear localization of Rtg1/3
occurs when cells are treated with rapamycin (9), which also results in
changes in Rtg3 phosphorylation levels (9). Although consensus remains
to be established on whether nuclear localization of Rtg3 occurs with
the hyperphosphorylated form, hypophosphorylated form, or both,
correlations between differences in transcription factor phosphorylation and retrograde gene expression are convincing (9, 26, 28, 33). Rtg2 is required for Rtg1/3 to mediate retrograde gene expression, and Mks1, originally thought also to be
required for Rtg1/3-mediated transcription (6, 8, 9), is now shown to
be a strong negative regulator of Rtg1/3-mediated transcription (24,
26, 33).
-ketoglutarate, the carbon skeleton of glutamate. More
important, however, we also found that Gln3 is not uniformly
distributed in the cytoplasm during growth in glutamine medium. It
appears to be sequestered in or associated with globular/tubular
structures. The implications of this distribution generate an
alternative way of viewing how Tor influences Gln3 intracellular
distribution and NCR-sensitive transcription.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of nitrogen source on intracellular
localization of Gln3 in carbon-starved cells grown in SC media.
Yeast cultures (TB123) were grown to mid-log in 2% glucose
synthetic complete (SC) or 2% glucose synthetic complete
with 0.1% glutamine replacing ammonia, the normal nitrogen source in
SC (SC + Gln), and transferred to the same media without
glucose. Aliquots were removed just prior to carbon starvation (0 min)
or at times following transfer to glucose-free medium and processed for
immunolocalization. Top panel, micrographs of cells grown in
SC (A and B) or SC + glutamine medium
(C and D) prior to (A and
C) and 180 min after the onset of carbon starvation
(B and D). Bottom panel, percentage of
the total yeast cells in which Gln3 was localized to the nucleus at
various times after transfer to carbon starvation medium.
View larger version (84K):
[in a new window]
Fig. 2.
Effect of ammonia or glutamine on
intracellular localization of Gln3 in carbon-starved cells grown in YNB
media. The experiment was performed as in Fig. 1 except for
changes in the media. Top panel, micrographs of cells after
180 min of incubation in YNB-glucose-free ammonium sulfate
(A) or YNB-glucose-free glutamine (B) media.
Bottom panel, as in Fig. 1.
View larger version (78K):
[in a new window]
Fig. 3.
Effect of nitrogen source on the
transcription of selected "control" and NCR-sensitive
(DAL5, DAL80) genes. Yeast
cultures (JK9-3da) were grown to mid-log in 2% glucose, YNB (without
ammonium sulfate and amino acids) and either 0.1% ammonium sulfate
(+NH4) or 0.1% glutamine (GLN)
as nitrogen source. Alternatively, cells were grown in SC medium.
Cultures were transferred to the same medium but devoid of glucose, and
samples were removed prior to (0 min) or at times following transfer to
glucose-free media. RNA blots were hybridized with
32P-radiolabeled DAL5 (allantoate permease)
(panel A), H3 (Hht1-histone3) (panels A and
C), ACT1 (actin) (panel B),
DAL80 (GATA-family transcription factor) (panel
B), and TCM1/RPL3 (a ribosomal protein) probes. PC4 (a
control used for sporulation assays whose expression mirrors ribosomal
RNA concentration) was used as a control.
View larger version (90K):
[in a new window]
Fig. 4.
Effect of nitrogen source on the
transcription of the GDH2 and NRG1 genes. The experiment was
performed as in Fig. 3.
-ketoglutarate to become limiting
in carbon starvation than glutamine in a glutamine to proline shift
makes reasonable physiological sense. We noted an additional
correlation with the glutamine to proline transition. The fraction of
cells in which Gln3 is nuclear increases sharply between 3 and 6 min,
plateaus between 6 and 15 min, and again rises sharply between 15 and
30 min. The cytoplasm of cells from the 30-min sample exhibited much
less fluorescence than at 15 min, arguing that nuclear localization of
Gln3 became more pronounced after 15 min (data not shown).
View larger version (41K):
[in a new window]
Fig. 5.
Comparison of the time courses of Gln3
movement into nuclei of cells transferred from glutamine to proline
medium versus YNB-ammonia to glucose-free YNB-ammonia
medium. The format of the experiment was as described in Fig.
1.
View larger version (42K):
[in a new window]
Fig. 6.
Comparison of the time courses of Gln3
movement into nuclei of cells transferred from YNB-glutamine to
YNB-proline medium versus YNB-ammonia to YNB-ammonia + rapamycin versus YNB-glutamine to YNB-glutamine + rapamycin media. The format of the experiment was as described for
Fig. 1, including those media containing rapamycin.
View larger version (27K):
[in a new window]
Fig. 7.
Intracellular distribution of Gln3 under
various growth conditions, visualized by conventional and deconvolution
microscopy. Yeast cultures (TB123) were grown to mid-log phase in
YNB-glutamine medium and processed for immunolocalization (row
3). Alternatively, cultures were grown to mid-log phase in
YNB-proline and transferred to YNB-glutamine medium. Aliquots were
removed just prior to (row 1), and 1 min after (row
2) the transfer from proline to glutamine medium.
Images are presented in pairs: Micrographs A,
C, E, and G were imaged using a Zeiss
Axioplan 2 imaging microscope. 0.1-µm sections were collected as a
Z-stack, and one image from the center of the cell is shown.
Micrographs B, D, F, and H
show these same images deconvolved with AxioVision 3.0 (Zeiss) software
using the constrained iterative algorithm.
View larger version (23K):
[in a new window]
Fig. 8.
TB123 cells were grown to mid-log phase in
YNB-glutamine medium and processed for immunolocalization.
Micrographs A-D were imaged using a Zeiss Axioplan 2 imaging microscope. 0.1-µm sections were collected as a Z-stack, and
one image from the center of the cell is shown. The images are shown
either as raw data (A) or as deconvolved images
(B-D) of Gln3p-mediated (A, B, and
D) or of DAPI-mediated fluorescence (C and
D). Three-dimensional views (E-G) were produced
from 15 images in a two-dimensional Z-stack, spanning about 1.5 µm of
the center of a yeast cell using AxioVision Inside 4D (Zeiss) software.
Images were rendered using the maximum projection in which only pixels
of the highest intensity along the axis are displayed.
Three-dimensional images have been rotated on all three axes to provide
different views of Gln3 intracellular localization.
View larger version (19K):
[in a new window]
Fig. 9.
The three-dimensional image described in Fig.
8 was rendered using the surface mode in which non-transparent surfaces
are calculated from gray values.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-ketoglutarate) become increasingly limiting, thereby slowing ammonia assimilation with concomitant loss of NCR and movement of Gln3
into the nucleus.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 901-448-6179;
Fax: 901-448-3244; E-mail: tcooper@utmem.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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