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Fernando Cardona
Agustn Aranda
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Yeast
Yeast 2009; 26: 115.
Published online in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/yea.1645
Research Article
*Correspondence to:
A. Aranda, Departament de
Bioqumica i Biologia Molecular,
Universitat de Val`encia, Apartado
73, Burjassot, Valencia
46100, Spain.
E-mail: agustin.aranda@uv.es
# Present address: Unitat de
Gen`etica Molecular, Institut de
Biomedicina de Val`encia (CSIC),
Valencia, Spain.
Abstract
Rsp5p is an essential ubiquitin ligase involved in many different cellular events,
including amino acid transporters degradation, transcription initiation and mRNA
export. It plays important role in both stress resistance and adaptation to the change
of nutrients. We have found that ubiquitination machinery is necessary for the correct
induction of the stress response SPI1 gene at the entry of the stationary phase. SPI1
is a gene whose expression is regulated by the nutritional status of the cell and whose
deletion causes hypersensitivity to various stresses, such as heat shock, alkaline stress
and oxidative stress. Its regulation is mastered by Rsp5p, as mutations in this gene
lead to a lower SPI1 expression. In this process, Rsp5p is helped by several proteins,
such as Rsp5p-interacting proteins Bul1p/2p, the ubiquitin conjugating protein Ubc1p
and ubiquitin proteases Ubp4p and Ubp16p. Moreover, a mutation in the RSP5 gene
has a global effect at the gene expression level when cells enter the stationary phase.
Rsp5p particularly controls the levels of the ribosomal proteins mRNAs at this stage.
Rsp5p is also necessary for a correct induction of p-bodies under stress conditions,
indicating that this protein plays an important role in the post-transcriptional fate of
mRNA under nutrient starvation. Copyright 2009 John Wiley & Sons, Ltd.
Keywords:
Introduction
Nutrient depletion in Saccharomyces cerevisiae
induces a plethora of physiological, biochemical
and morphological changes. A yeast culture grown
on glucose-based medium changes from a fermenting metabolism to a respiratory one when glucose
becomes limited. This phenomenon is called the
diauxic shift. When the carbon source is exhausted,
the culture enters the so-called stationary phase
(Gray et al., 2004; Herman, 2002). This state
allows cell survival over long periods of time without added nutrients. Cells in stationary phase cultures accumulate glycogen and trehalose, develop a
thickened cell wall and become resistant to stresses
such as increased temperature and oxidative stress.
Copyright 2009 John Wiley & Sons, Ltd.
Description
Origin
EUROSCARF
EUROSCARF
F. Abe
F. Abe
F. Abe
F. Abe
This work
This work
This work
This work
This work
This work
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
R. Rothstein
B. Andre
B. Andre
B. Andre
J. Heitman
J. Heitman
stationary phase and that it has a positive role in pbody formation in the glucose-depleted condition.
-galactosidase analysis
The SPI1placZ fusion expression was determined
as -galactosidase activity via the method of permeabilized cells, using ONPG as a substrate, as
described by Adams et al. (1997). To detect blue
colonies, plates containing SC ura + Xgal
0.4 mg/ml + phosphate buffer, pH 7, were used.
The white colonies defective in SPI1placZ fusion
activity were transformed with a library constructed
in the episomal plasmid YEp32 (a gift from J. C.
Igual). The transformation was replica-plated on Xgal plates.
RNA analysis
RNA isolation, quantification and analysis were
carried out as previously described (Carrasco et al.,
2003). The expression of the SPI1 gene on several
strains and conditions was followed by Northern
blot analysis, using a specific probe for this gene
obtained with the oligonucleotides SPI1-F/G (see
Supporting information, Table S1).
For microarray analyses, cDNA preparation,
labelling and hybridization were carried out as
described by Fazzio et al. (2001). The combinations for hybridization Cy3Cy5 were: WTa
HPG11a, HPG11bWTb, where the a and b
samples were obtained from two independent cultures of each strain. The intensity obtained in
each channel for each pair of microarrays was
normalized by Lowess (Yang et al., 2002). The
overrepresentation of categories containing functionally related genes in each strain was statistically analysed using the Function Associate
tool (http://llama.med.harvard.edu/cgi/func/func
associate). The analysis of the transcription factors involved in the expression of differentially
expressed genes was carried out with the YEASTRACT package (http://www.yeastracts.com).
Copyright 2009 John Wiley & Sons, Ltd.
The expression of some genes with the differential expression in the wild-type (WT) and HPG1-1
strains in the microarray analysis was also analysed
by semiquantitative RTPCR. cDNA preparation
was carried out as previously described (JimenezMart et al., 2007). For the amplification of specific
genes in the resulting cDNA, it was diluted 20-fold
and 5 l were used for a PCR reaction carried out in
a final volume of 1525 l, containing the primers
(0.5 mM of each), dNTPs (0.2 mM of each), MgCl2
(3 mM), buffer and 1 U DNA polymerase Biotaq
(Bioline) from Thermus aquaticus YT-1. The reaction conditions were: 1 cycle at 94 C for 3 min,
2025 cycles at 94 C for 1 min, 1 min at the optimal primer hybridization temperature in each case,
1 min at 72 C and, finally, a 10 min cycle at 72 C.
The ACT1 gene was used to normalize the data.
The specific oligonucleotides used for the analysis
of each gene considered are shown in the Supporting information.
Western blotting
To prepare protein extracts, 2.5 OD unit cells
were collected by centrifugation and were resuspended in 100 l water, then 100 l NaOH 0.2
M were added and the preparation was incubated
for 5 min. After centrifuging at 12 000 r.p.m.
for 1 min, the pellet was resuspended in 50 l 2
loading buffer (TrisHCl 150 mM, pH 6.8, DTT
300 mM, SDS 6%, bromophenol blue 0.3%, glycerol 30%) and incubated at 95 C for 5 min. After
cooling and centrifuging (3000 r.p.m. for 10 min),
the supernatant was collected. The total protein
concentration was determined by the Bradford
method (BioRad), and 50 g protein was used for
each analysis. After SDSPAGE electrophoresis
in 7.5% polyacrylamide, proteins were transferred
to a 0.45 m nitrocellulose membrane (Trans-Blot,
1620113, Bio-Rad) by electrotransference. Immunodetection was carried out using a GFP antibody
(Sigma). This primary antibody and the secondary
antibody (peroxidase anti-rabbit) were diluted at
1/10 000 and 1/50 000, respectively. Blocking and
incubation with the antibodies were carried out
in TBST buffer (20 mM TrisHCl, pH 8, 0.5 M
NaCl, 0.05% v/v Tween 20) with 5% w/v non-fat
dried milk. Washes were done in TBST. ECF plus
the Western Blotting detection system (RPN2132,
Amersham) were used for detection, following the
manufacturers instructions.
Yeast 2009; 26: 115.
DOI: 10.1002/yea
N/No
Results
160
140
120
100
80
60
40
20
0
BY4742
spi1
10
15
20 25
t (days)
30
35
40
45
this gene during growth is around 7 h after the culture started, with a dilution of an overnight culture
at OD600 = 0.5. Similar results were observed in
the rich medium YPD using microarrays (Gasch
et al., 2000), where the maximum of expression is
reached between 612 h (depending on individual
experiments) from a starting point of OD = 0.3,
and its expression remains high throughout the
stationary phase. However, in our -galactosidase
assay, the enzyme activity decreases at later stages
of growth, probably reflecting the general decrease
in translation that happens in the stationary phase
(Gray et al., 2004). Therefore, the SPI1placZ
construct is a useful tool to study the gene expression changes previous to the entry into the stationary phase.
Table 2. Stress tolerance in the wild-type and spi1 mutant. The percentage of colony-forming units after the stress
condition is shown
wt
spi1
Ethanol
Heat shock
Low pH
High pH
Oxidative (mm)
22 1.5%
17 1.2%
0.88 0.001%
0.42 0.0001%
67 7%
56 2%
25 2%
10 1%
3.08 0.15
3.9 0.3
For oxidative stress, the diameter (mm) of the inhibition halo is shown. All experiments were carried out in triplicate. SD is shown.
Copyright 2009 John Wiley & Sons, Ltd.
-galactosidase (a.u)
35
WT
HPG1-1
30
25
20
15
10
5
0
0
12
16
20
24
time (hours)
B
m4
pTRP1
YPH499
E PD
pTAT2
HPG1-1
E PD
SPI1
rRNA
-galactosidase (a.u.)
30
WT
npi1
25
20
15
10
5
0
12
16
20
24
12
16
time (hours)
20
24
time (hours)
-galactosidase (a.u.)
-galactosidase (a.u.)
40
35
30
25
20
15
10
5
0
WT
bul1bul2
2
1.5
1
0.5
sok2
ubp14
ubp6
ubp4
ubc7
ubc5
ubc4
ubc2
ubc1
ubi4
WT
in the postdiauxic phase (Figure 3C). S. cerevisiae ubiquitin is a 76-amino acid protein encoded
by four structural genes, UBI1, UBI2, UBI3 and
UBI4. While each of these genes is reported to
be expressed during yeast exponential growth, only
Copyright 2009 John Wiley & Sons, Ltd.
(A) Induced
Categories
Genes
Translation
Cell wall
Other
(B) Repressed
Amino acid
metabolism
Proline transport
Stress
Table 3. Continued
Categories
Other
Unknown
Genes
PHO89 (4.73), FMP45 (4.36), FMP16 (4.11),
NDE2 (3.73), NQM1 (3.63), SPS100, (3.57),
PHM6 (2.95), YCT1 (2.93), GND2 (2.88),
MSC1 (2.78), AQY1 (2.71), PUR5 (2.67),
NCE102 (2.60), FRM2 (2.58), GMP2 (2.40),
LAP4 (2.30), BOP2 (2.26), PHM7 (2.20),
PDH1 (2.17), ALD4 (2.15), FLR1 (2.06),
ZRT1 (2.04), RMD6 (2.03)
YMR107w (6.93), YDL218w (5.04),
YKL071w (2.82), YHR033w (2.60),
YHR140w (2.48), YBR285w (2.32), YAL061w
(2.24), YGR154c (2.16), YDL223c (2.10)
Those genes that are induced or repressed by >2 are shown, and
their induction or repression values are shown in brackets.
WT
HPG1-1
RPL9a
CHA1
GAS3
SPI1
HSP26
SER3
MET3
GAP1
ACT1
Figure 5. Effects of toxic agents on the growth of ubiquitination mutants. The HPG1-1, HPG1-4 and bul1bul2 mutants
were replica-plated on (A) SDS 0.008% or rapamycin 0.1 g/ml and (B) cycloheximide 0.1 g/ml
Copyright 2009 John Wiley & Sons, Ltd.
10
bul1bul2 Dcp2p-GFP
HPG1-1 Dcp2p-GFP
A WT Dcp2p-GFP
YP 15
1M KCl 15
HPG1-1 Dhh1p-GFP
WT Dhh1p-GFP
bul1bul2 Dhh1p-GFP
YP 15
1M KCl 15
bul1bul2
HPG1-1
YP / 15
WT
bul1bul2
HPG1-1
KCl 1M / 15
WT
Dcp2p-GFP
Ponceau
Dhh1p-GFP
Ponceau
Figure 6. The RSP5 mutant is defective in p-body formation. Microscopy after 15 min without glucose (YP) or with osmotic
stress (1 M KCl) in WT, HPG1-1 and bul1bul2 strains transformed with (A) Dcp2pGFP fusion and (B) Dhh1pGFP
fusion. (C) Western blot of the Dcp2pGFP and Dhh1pGFP protein levels of these strains under the same stress
conditions, using an anti-GFP antibody
protein kinase that links nutrient status and cell proliferation, target of rapamycin (TOR). Our mutations were hypersensitive to rapamycin, indicating
that ubiquitination machinery contributes to the
response to nutrient starvation.
A relationship between Rsp5p and translation
accuracy has been recently reported (Kwapisz
et al., 2006), according to which a S. cerevisiae
strain carrying an rsp5-13 mutation shows altered
sensitivity to antibiotics and a slower rate of translation. The results obtained in the microarray and
in the semi-quantitative RTPCR analysis indicate that there is a higher level of transcripts
related to protein biosynthesis in the mutant HPG11 under stationary phase conditions. In order to
confirm this effect on the translation of the rsp5
mutant alleles that we used and the role of the
BUL1/2 role in translation, experiments of resistance to the translation inhibitor cycloheximide
were carried out (Figure 5B). The HPG1-1 allele
presented a significant defect on translation. However, the HPG1-4 allele did not have such a significant growth defect. Therefore, the cycloheximide sensitivity showed some allele specificity.
The bul1/2 deletion also revealed an increased
cycloheximide sensitivity. These data confirm the
role of Rsp5pBul1pBul2p in translation and
indicate that this role may be related to the alteration of the translation machinery expression.
11
Rsp5p plays a role in gene expression at the posttranscriptional level (see Introduction). Therefore
we tested the effect of our ubiquitination mutants in
another post-transcriptional event related to stress
and nutrient starvation, that of the formation of processing bodies (p-bodies).
To analyse this event, we fused the green fluorescent protein GFP to two proteins implicated
in mRNA degradation that are well established pbody markers, Dcp2p and Dhh1p in the HPG1-1
and the bul1/bul2 mutants. Under glucose deprivation stress (15 min in YP) and osmotic shock
(15 min in 1 M KCl), the p-bodies were clearly
formed in the wild-type cells, both when Dcp2p
(Figure 6A) and Dhh1p (Figure 6B) were fused to
GFP. In the RSP5 mutant tested, HPG1-1, the number and intensity of the p-bodies decreased for both
GFP fusions (Figure 6) in both conditions, particularly in the starvation condition. This effect was not
seen in the bul1/bul2 mutant, where there is still
a high number of cells containing p-bodies after
the stress. Therefore, the Rsp5p has an important
role in the formation of p-bodies under different
stress conditions without the help of Bul1/2p in
this particular event.
In order to rule out a defect in the fusion protein amount, we carried out a Western blot analysis using an anti-GFP antibody (Figure 6C). The
levels of GFP-fused Dcp2p and Dhh1p proteins
were similar in the HPG1-1 mutant compared to
the wild-type. Therefore, Rsp5 seems to affect the
assembly of the p-bodies during these environmental changes, not their protein levels.
Discussion
In this work, we have used the SPI1 gene as a
marker of the entry into the stationary phase. Spi1p
is a GPI-anchored cell wall protein that plays a
positive role in stress response. Its deletion caused
an increased sensitivity to various insults, such as
heat shock, high ethanol concentration, extreme pH
values, oxidative stress and starvation (Table 2).
As a typical stress-regulated gene, SPI1 expression is induced under many adverse conditions
(Gasch et al., 2000) and is highly expressed in the
late growth phases under both laboratory (Gasch
et al., 2000) and industrial conditions (Puig and
Perez-Ortn, 2000). We have constructed a fusion
of the SPI1 promoter to lacZ to identify new
Yeast 2009; 26: 115.
DOI: 10.1002/yea
12
13
Acknowledgements
We are indebted to F. Estruch for supplying us with the
GFP antibody and J. C. Igual for the multicopy library.
We also thank F. Abe, B. Andre and J. Heitman for the
strains provided. This work was supported by Grant No.
AGL2005-00508 from the Ministerio de Educacion y Ciencia and Grant No. GRUPOS03/012 from the Generalitat
Valenciana to M.O. and A.A. F.C. was an FPU Fellow of
the Ministerio de Educacion y Ciencia.
Supporting Information
Supporting information may be found in the online
version of this article.
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