Protein & Peptide Letters, 2010, 17, 1489-1494
1489
Quantitative Effects of Magnesium Chloride Stress on Aggregation of
Sup35p in [psi-] Yeast Cells
Yao Song1, Wanjun Lan1, Xianyuan Wu1, Jianwei He1,2, Hui Li1,2, Songbin Ben1 and
Youtao Song1,2*
1
School of Life Science, Liaoning University, Shenyang 110036, China; 2Province Key Laboratory of Animal Resource
and Epidemic Disease Prevention, Shenyang 110036, China
Abstract: [PSI+] phenotype can be transiently induced when Magnesium chloride (MgCl2) was the selective pressure in
SUP35 repeat-expansion mutant [psi-] yeast strains. We further investigated [PSI+ ] phenotype change under different
MgCl2 conditions with native Sup35p and quantified the Sup35p status changes with fluorescence recovery after photobleaching (FRAP) and semi-denaturing detergent-agarose gel electrophoresis (SDD-AGE) analysis. It was found that
the [PSI+ ] phenotype presented a dose-dependent relationship with the concentrations of MgCl2. Furthermore, Sup35p aggregated in MgCl2 treated cells but did not form large aggregates as it does in [PSI+] cells, and the size of Sup35p aggregates showed a time-dependent relationship with the MgCl2 application. The aggregation of Sup35p strictly depended on
the presence of MgCl2 stress in our strains.
Keywords: Magnesium chloride, prion, [PSI+], quantitative effects, Sup35p, stress.
INTRODUCTION
+
[PSI ] was first identified in Saccharomyces cerevisiae as
a cytoplasmic genetic determinant that modulates the efficiency of nonsense suppression and was subsequently shown
to be the prion form of the translation termination factor
Sup35p [1, 2]. Sup35p contains three sub-domains: a dispensable, polar amino acids-rich amino-terminal domain (N)
that is essential for Sup35p prion formation; a dispensable
highly charged middle domain (M) modulating [PSI+]
propagation; and a C-terminal domain (C) that is necessary
and sufficient for Sup35p to function as a release factor [3,
4]. Sup35p becomes inoperative for translation termination
when its N terminus switches to an aggregating amyloid conformation [5]. When the appearance of [PSI+] caused readthrough translation, previously cryptic genetic variations
were revealed and enhanced the rate of evolution in these
cells [6, 7]. Recent research on environmental stress to yeast
prion [PSI+] phenotype change has found that 1.0 M MgCl2
induces [psi-] to [PSI+] phenotype in an SUP35 repeatexpansion mutant strain but the effect of MgCl2 on [PSI+]
induction was very low and was not statistically significant
[7]. However, these observations were limited to phenotype
studies, and there has been little information about the status
of Sup35p changes in the [psi-] strain with native Sup35p
when cells are stressed by MgCl2 at the cellular and protein
levels.
Traditionally, aggregation status analysis of Sup35p in
yeast cells has been realized by running the pellet fraction
(insoluble polymers) and the supernatant fraction (soluble
monomers) through SDS-PAGE and followed by im*Address correspondence to this author at the School of Life Science,
Liaoning University, Shenyang 110036, China; Tel: 86-24-6220-2280; Fax:
86-24-6220-2280; E-mail: [email protected]
0929-8665/10 $55.00+.00
munoblotting [8, 9]. However, this SDS-denatured method
cannot detect the relative size of polymers which are SDSresistant and play an important role in the aggregation of
Sup35p. Semi-denaturing detergent-agarose gel electrophoresis (SDD-AGE) allows the collection of more molecular
weight information and better quantifies the prion aggregation properties than SDS-PAGE does [10]. In our previous
research, we further improved SDD-AGE to make the process faster and the manipulation easier [11].
Fusion protein technology with Green Fluorescent Protein (GFP) is widely used for real-time analysis of molecular
events in living cells. We previously found that GFP fusion
proteins inserted between the N and M domains of Sup35p
(NGMC) function normally in translation termination and
[PSI+] propagation [11]. This NGMC expression makes it
possible that Sup35p aggregates (foci) can be visualized by
GFP fluorescence. Furthermore, the fluorescence recovery
after photobleaching (FRAP) is a non-invasive method to
examine protein motility and aggregation in real-time in the
living cell, and it can be used to quantify the extent of aggregation using a confocal microscope, which provides a more
sensitive technique to detect [PSI+] aggregates in yeast cells
[11, 12]. This method enables us to detect quantitative differences in mobility of the GFP proteins which are diffusing
freely in the cell.
Besides the phenotype assay, the two techniques mentioned above were used in this research to quantify the status
of Sup35p aggregation when cells were in the MgCl2 stress
condition. The information obtained from these results not
only quantifies the effects of MgCl2 stress condition on
[PSI+] formation, but also sheds light on the experimental
methods to quantify stress-induced [PSI+] formation at the
cellular and the protein levels.
© 2010 Bentham Science Publishers Ltd.
1490 Protein & Peptide Letters, 2010, Vol. 17, No. 12
MATERIALS AND METHODS
Strains, Media, and Growing Conditions
Strains 779-6A (MAT kar1 SUQ5 ade2-1 his3202
leu21 trp163 ura3-52, [PSI+]) and 780-1D (MAT
sup35::KanMX /pJ510, [PSI+]) were used in our experiments
[11]. The [psi-] variants of these strains were obtained by 5
mM guanidine curing. The concentration of adenine (~10
mg/l) in the media allowed ade2-1 cells to grow but was not
high enough to repress the adenine biosynthetic pathway. In
ade2-1 cells, a pigmented adenine precursor accumulates
under these conditions because of a block in the pathway. 1/2
YPD (composed of 0.5% yeast extract, 2% peptone, and 2%
glucose) is a complex medium with a limited but undefined
amount of adenine. YPAD (excess adenine) contains 1%
yeast extract, 2% peptone, 2% glucose and 400 mg/liter adenine. Synthetic media was as described [13], and solid media
contained 2% agar. Cells were grown at 30°C unless indicated.
Microscopy
Microscopic analysis of the NGMC fusion proteins was
done with an Olympus FV1000S-SIM/IX81 microscope at
China Medical University’s Laboratory Technology and Experimental Medicine Center. Yeast cells were incubated in
YPAD liquid medium or YPAD liquid medium contained 1
M MgCl2. Fluorescent images were captured with FV10ASW 1.6 Viewer software and processed with Adobe Photoshop software.
Confocal Microscopy and Fluorescence Recovery after
Photobleaching (FRAP)
FRAP assay was done with an Olympus FV1000SSIM/IX81 confocal microscope and was slightly modified as
described [11, 12]. Yeast cells were incubated in YPAD liquid medium or YPAD liquid medium contained 1 M MgCl2
for the indicated number of days. When data sets were prepared, the cells were photobleached and monitored identically including the number of bleaches, the area of the photobleach region, and the time course of imaging at low laser
power.
Semi-Denaturing Detergent-Agarose Gel Electrophoresis
(SDD-AGE) and Western Blot Analysis
A previously described method for agarose gel electrophoresis was modified in this study [11]. Cell lysates were
incubated in a sample buffer of (Tris-borate-EDTA, 0.5%
SDS, 5% glycerol, and 0.05% bromophenol blue) for 10 min
at 42°C, and then the proteins were separated by electrophoresis in horizontal 1.2% agarose gels (15 by 15 cm) in Trisborate-EDTA buffer consisting of 0.1% SDS. Proteins were
electrophoretically transferred to Immo-bilon polyvinylidene
difluoride sheets (Millipore) at 100 V for 60 min at 4°C, and
Western analysis was performed as described [9].
RESULTS
Effects of Different Concentrations of MgCl2 on [PSI+]
Phenotype Characteristics in Wild-Type [psi–] Cells
In [PSI+] cells most Sup35p is in aggregated form, which
reduces translation termination efficiency and causes non-
Song et al.
sense suppression. The presence of [PSI+] is monitored by its
ability to suppress the ade2-1 nonsense allele when the
SUQ5 tRNA is present [1]. Nonsuppressed ade2-1 mutants
require adenine and are red in color due to the accumulation
of the Ade2p substrate, while [PSI+] (Ade+ phenotype) restores adenine prototrophy and the colony color turns to
white [1]. This coloration assay was used to test effects of
different MgCl2 stress condition on [PSI+] induction. Fig.
(1A) shows that the phenotype of wild-type yeast [psi–]
colonies changed and trended toward the Ade+ phenotype
when the indicated concentrations of MgCl2 were added to
the medium. The cells started to grow slowly on plates containing 1 M MgCl2, and the effects of 1.5 M MgCl2 on Ade+
phenotype induction could not be detected because 1.5 M
MgCl2 strongly inhibited yeast cell growth. The coloration of
[psi–] colonies shifted from red to pink, and even white on
1/2YPD medium with 0.05, 0.25, 0.5, 1 and 1.5 M MgCl2
(Fig. 1A, row 1 and 2). Correspondingly, the [psi–] cells began growing on the SD-Ade mediums containing the indicated concentrations of MgCl2 (Fig. 1A, row 3). Interestingly, red colonies were present on SD-Ade medium containing MgCl2, in addition to several irregular white colonies.
This result suggests that the Ade+ colonies induced by MgCl2
might be a weakened [PSI+] phenotype. This phenotype shift
reflected the suppression of ade2-1 from least to most, indicating that the Ade+ phenotype became gradually stronger.
These results suggest a novel dose-dependent relationship
between the MgCl2 concentrations and the Ade+ phenotype
change. Moreover, as shown in Fig. (1B), the coloration of
MgCl2 induced yeast colonies turned back to red on 1/2YPD
medium and red colonies which presented on SD-Ade mediums containing MgCl2 did not grow on SD-Ade mediums
after MgCl2 was removed. Although some irregular white
colonies appeared on SD-Ade plates, the results of guanidine-hydrochloride curing showed that all these colonies
were pseudo positive. These white colonies might be due to
mutations induced by MgCl2 stress. Fig. (1C) shows parts of
the representative data. Together, these results suggest
MgCl2 dose increasing could result in different intensity of
the Ade+ phenotype, but the propagation of the induced Ade+
phenotype strictly depended on the MgCl2 stress condition.
However, the Ade+ phenotype may simply be a result of less
efficient stop codon recognition under high MgCl2 concentrations. To further evaluate this nonsense suppression,
which might be led by Sup35p status changes in MgCl2
treated cells, methodologically sophisticated studies are required.
Live-Time Fluorescence Analysis of [psi–] Cells Under the
MgCl2 Stress Condition
With the expression of Sup35p-GFP fusion NGMC constructed in our previous research, the aggregation state of
Sup35p was monitored in living cells with fluorescence microscopy [11]. In stationary-phase [GPSI+] cells, fluorescent
foci were detected in the cytoplasm, whereas in log-phase
[GPSI+] cells and [Gpsi–] cells, GFP fluorescence was diffusely present throughout the cytoplasm [11]. Thus, this
fluorescent assay was used to detect the [PSI+] status as a
supplementary method to the coloration assay. Unexpectedly, the GFP fluorescence in cells induced with 1 M MgCl2
was diffusely present not only during log-phase (1 day) but
Effects of MgCl2 Stress on Sup35p
Protein & Peptide Letters, 2010, Vol. 17, No. 12
1491
Figure 1. Effects of different MgCl2 levels on the [PSI+] phenotype. (A) [psi–] cells were first grown as colonies on 1/2 YPD for 2 days at
30°C, then the cells were replica plated onto indicated concentrations of MgCl2 contained 1/2 YPD and SD-Ade plates. The plates were further incubated for 3 days at 30°C, and then 5 days at 4°C. (B) Colonies on 1/2 YPD plates containing indicated concentrations of MgCl2
(panel A, second row) were replicated onto 1/2YPD, SD-Ade and non MgCl2 YPAD plates, which were further incubated for 3 days at 30°C,
and then 5 days at 4°C. On YPAD medium, all the cells presented pure white, which suggested that the red cells on 1/2 YPD plates were
really [psi–]. The last column is was [PSI+] cells streaked on indicated plates as control. (C) White colonies on SD-Ade plates marked with
lowercase letters (panel B, second row) were streaked onto 1/2YPD medium supplemented with 5 mM guanidine and incubated for 3 days at
30°C, and then 5 days at 4°C. Due to space limitations, parts of the representative data were showed although all of the white colonies were
tested. + cells were streaked onto the same plates as control. The experiments were repeated 3 times; each time yielded similar results.
also stationary-phase (2 or 3 days) throughout the cytoplasm
(Fig. 2), which was unparallel with the coloration phenotypic
assay. This result suggested that the aggregates of Sup35p in
MgCl2 induced [PSI+] phenotype cells might not be large
enough to be detected as fluorescent foci.
To further study the actual status of Sup35p in MgCl2
induced [PSI+] phenotype cells, we measured fluorescence
recovery after photobleaching to detect the size of NGMC
aggregates in the 1 M MgCl2 treated cells. Since the diffusion rate of particles is related to particle mass, the time required for the recovery of fluorescence by diffusion of
unbleached protein after photobleaching would be longer for
more massive particles. Compared to isogenic [Gpsi–] cells,
there was a significant increase in recovery time for MgCl2
treated [Gpsi–] cells, and the recovery time increased in proportion to the MgCl2 treatment time (Fig. 3A). This result
suggested that NGMC had aggregated in such cells. When
the MgCl2 application days increased, the recovery time also
increased and was closer to that of log-phase [GPSI+], so a
time-dependent relationship was presented. Unfortunately,
by the 4th day the cells were mostly dead and the fluorescence signal in our strain was too feeble (<1, 000 unit) to be
detected by this assay. On the other hand, when MgCl2 was
removed from the culture medium the recovery time decreased significantly and was close to the level of [psi–]. Fig.
(3B) indicating that the aggregated NGMC might be dissolved. These results suggested that the propagation of the
induced [PSI+] strictly depends on the MgCl2 stress conditions, which is also consistent with the above phenotypic
assay.
Semi-Denaturing Detergent-Agarose gel Electrophoresis
(SDD-AGE) Analysis of Wild-Type [psi–] Cells Under the
MgCl2 Stress Condition
Previous research has shown that SDD-AGE is an efficient method to detect the status of SDS-insoluble Sup35p
polymers which might act as “seeds” [10, 11, 14]. Therefore,
in order to validate the phenomenon observed at the protein
level during the FRAP experiment, we further performed an
SDD-AGE assay to study these inconspicuous “[PSI+]” cells
1492 Protein & Peptide Letters, 2010, Vol. 17, No. 12
Song et al.
gregates in MgCl2 induced cells, (iii) the aggregation of
Sup35p presented a time-dependent relationship with application of MgCl2, and (iv) the appearance of Sup35p aggregates strictly depended on the presence of MgCl2. The results
obtained in this work that Sup35p aggregated under the
MgCl2 stress condition and dissolved after the stress removed, have provided some evidence to confirm the hypothesis that [PSI+] represents a mechanism for evolvability
at the cellular and protein levels.
Figure 2. Effects of 1 M MgCl2 on [Gpsi–] cells with fluorescence
foci assay. [Gpsi–] cells were kept in YPAD liquid medium containing 1 M MgCl2 at 30. At days 1, 2, and 3 the bacterial suspension
was diluted with sterile water then observed under microscopy and
fluorescent images were captured. [GPSI+] and [Gpsi-] cells were
incubated in the same conditions as the positive and negative control but medium did not contain any extra compounds.
induced by MgCl2. [psi–] cells were incubated in 1/2YPD
liquid culture with 1 M MgCl2 for 1-3 days and then collected by centrifugation. After fractionating the cell lysates
on 1.2% agarose gel followed by immunoblot with rabbit
antiserum raised against Sup35p, we found that the size distribution of Sup35p from MgCl2 treated [psi–] cells significantly increased with increased culture time (Fig. 4, 1d, 2d,
3d). SDS-insoluble Sup35p polymers were detected in
MgCl2 treated [psi–] cells and the size of the polymers increased in a time dependent manner. However, these SDSresistant Sup35p polymers were apparently smaller than
those polymers in [PSI+] cells. Moreover, samples cultured
with mediums deprived of MgCl2 were also investigated
using this assay. As shown in Fig. 4 (1d’, 2d’, 3d’), there
was no significant difference in mobility between cells with
the MgCl2 removed and our control [psi–] cells, which indicated SDS-insoluble Sup35p polymers disappeared after
removal of the MgCl2. These results suggested that the generation of “[PSI+]” cells in our study were strictly dependent
on the level of MgCl2 stress condition, which is consistent
with the phenotypic changes and FRAP measurements.
Figure 3. Diffusion and dynamics of NGMC in 1 M MgCl2 treated
[Gpsi–] cells detected by FRAP. Recovery of fluorescence into photobleached areas of cells was monitored as a function of time.
FRAP of NGMC in yeast cells was performed as described in the
methods. (A) Dynamics of NGMC diffusion in [Gpsi–] cells treated
with 1 M MgCl2 for 1d, 2d and 3d. (B) FRAP of NGMC cells
(treated with MgCl2 as panel A) further incubated in YPAD medium devoid of MgCl2 for 1 more day (1d’, 2d’, 3d’). Data were
obtained using 15-20 cells for each FRAP experiment. The average
and standard deviation for each time point was calculated.
DISCUSSION
In this work, we conducted an in-depth study of effects of
MgCl2 stress on yeast prion [PSI+] in S. cerevisiae with native Sup35p, and established quantitative assays that were
composed of fluorescent foci, FRAP and SDD-AGE technique to study prion aggregation at the cellular and protein
levels. Our results showed that (i) the MgCl2 stress could
induce Ade+ phenotype appearance in a dose-dependent
manner, (ii) Sup35p aggregated but did not form large ag-
Recently, a series of studies have proposed that the assembly and disassembly of the prion amyloid are controlled
by a complex web of interactions between Sup35p and the
chaperone network of the cell have been shown to modify
the frequency of de novo prion formation in yeast [7, 15, 16].
Under the MgCl2 stress condition, the factors in the complex
network might change their metabolic behavior and thus
cause Sup35p aggregation. For instance, Fung et al. have
Effects of MgCl2 Stress on Sup35p
reported that MgCl2 affects the activity of yeast cytosolic
Hsp70 Ssa1p by enhancing ATPs ability to bind to the ATPase domain [17], which facilitates aggregation of Sup35p
and consequently produces the [PSI+] phenotype [18, 19].
Protein & Peptide Letters, 2010, Vol. 17, No. 12
1493
the influence of [PIN+] to Sup35p polymerization cannot be
readily excluded. To confirm our hypothesis further research
on detecting the Rnq1p status with anti-Rnq1p antibody and
GFP fusion in our [psi] strain is needed.
ACKNOWLEDGMENTS
This work is supported by the National Natural Science
Foundation of China (NSFC, grant No. 30600113,
No.30970125), partially sponsored by the Fund of Liaoning
Provincial Education Department (grant No. 2008S100, No.
2009R26). We are grateful to Daniel C. Masison (NIDDK,
National Institutes of Health, USA) for reading the manuscript and offering his constructive criticisms; Jian Gao
(China Medical University) for technical assistance in confocal microscopy operation; Wei Liu (University of California
Davis) and Andrew Coash (Liaoning University) for their
revisions of the manuscript.
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Figure 4. Size of SDS-resistant Sup35p polymers in MgCl2 treated
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