title: monitoring adp/atp ratio in yeast cells using the
TRANSCRIPT
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Title: Monitoring ADP/ATP ratio in yeast cells using the 1
fluorescent-protein reporter PercevalHR 2
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Authors: Phuong Thi Mai Nguyen, Yuki Ishiwata-Kimata, Yukio Kimata 4
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Affiliation: Graduate School of Science and Technology, Nara Institute of Science and 6
Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan 7
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Running head: Monitoring ADP/ATP ratio with fluorescent protein in yeast 9
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Corresponding author: Yukio Kimata 11
TEL 0743-72-5422 12
FAX 06-6537-1665 13
E-mail [email protected] 14
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Abstract 16
PercevalHR (Perceval High Resolution) is an artificially designed fluorescent protein, 17
which changes its excitation spectrum based on the ADP/ATP ratio of the environment. 18
Here we demonstrated that PercevalHR can be used for monitoring energy status of 19
Saccharomyces cerevisiae cells, which are affected by diauxic shift and mitochondria 20
inhibition, in a non-invasive and non-destructive manner. 21
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Keywords 23
Fluorescent reporter, ATP, ADP, yeast, diauxic shift 24
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Introduction 26
To date, a number of genetically encoded fluorescent biosensors have been developed 27
as reviewed in Ref. [1]. They are modified versions of fluorescent proteins and change 28
their fluorescence properties depending on specific surrounding conditions, such as pH, 29
redox, temperature and abundance of a specific small molecule or metal ion. When 30
genetically expressed in cells, such biosensors emit fluorescent signals, which can be 31
quantitatively detected using fluorescence microscopic or flowcytometric techniques. 32
This methodology allows researchers to monitor a specific intracellular status in a quick, 33
non-invasive and non-destructive manner. 34
Perceval is a fluorescent biosensor that has been artificially created through genetic 35
connection of fragments of the GFP variant Venus and the bacterial ATP/ADP-binding 36
protein GlnK1 [2]. While ADP and ATP are competitively captured by Perceval, its 37
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excitation spectrum is more affected by ATP than ADP because ATP leads to a drastic 38
conformational change of GlnK1. PercevalHR (High Resolution) is an improved version 39
of Perceval with higher sensitivity for ADP/ATP ratio [3]. The excitation spectrum of 40
PercevalHR has two peaks (500 nm and 420nm), and the ratio of the heights of these two 41
peaks varies depending on the ADP/ATP ratio of surrounding (longer-wavelength 42
excitation upon ATP binding). In contrast, the emission spectrum (peak at 520 nm) is 43
unchanged. Although PercevalHR was used for imaging energy status in mammalian cells 44
[3], to our knowledge, no report has shown usage of Perceval or PercevalHR in fungal 45
species including yeast cells. 46
Saccharomyces cerevisiae cells proliferate rapidly during the log phase in glucose-47
based media through the production of ATP from ADP by glycolysis, namely 48
fermentation, while mitochondrial respiration is induced upon exhaustion of glucose. This 49
switch from fermentation to aerobic respiration is known as diauxic shift and is 50
accompanied by a global change in the cellular status [4]. Possibly because of low 51
performance of the mitochondrial F1/F0-ATP synthase [5], S. cerevisiae cells produce 52
ATP less effectively and grow slowly in the respiratory phase. Thus, for physiological 53
and energetical studies of yeast cells including those on diauxic shift, monitoring 54
intracellular ADP/ATP ratio could provide important information. 55
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Materials and Methods 57
Plasmid and yeast strain: In the present study, the PercevalHR-coding sequence flanked 58
by the S. cerevisiae strong TDH3 promoter and CYC1 transcription terminator was cloned 59
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into pRS425, which is an S. cerevisiae YEp-type vector carrying the LEU2 selectable 60
marker [6]. The resulting plasmid, named as pRS425-PercevalHR, together with its 61
sequence information is now available in the National BioResource Project 62
(http://yeast.nig.ac.jp/yeast/top.xhtml; NBRP ID: BYP9646). 63
Throughout this study, we employed S. cerevisiae strain KMY1005 (MATa ura3 leu2 64
his3 trp1 lys2; Ref. [7]) carrying the indicated plasmids, and cultured it in standard 65
synthetic glucose (dextrose) medium (SD medium) supplemented with auxotrophic 66
requirements at 30 °C. Culture optical density (OD600) were monitored by using the 67
SmartSpec3000 spectrophotometer (BioRad). 68
Microscopy and image analysis: Cells were observed under the Keyence BZ-9000E 69
fluorescence microscope equipped with the objective lens CFI Plan Apo l100xH (Nikon). 70
As described in Ref. [8], florescence images were observed using the filter set OP-79301 71
(excitation wavelength, 472.5/30; dichroic mirror wavelength, 495; emission, 520/35) for 72
excitation with blue light (1.0-sec exposure) and a custom-made filter set (excitation 73
wavelength, 395/25; dichroic mirror wavelength, 495; emission, 510/20) for excitation 74
with UV/violet light (1.5-sec exposure). 75
To obtain quantified data from the microscopic analysis, approximately 4 to 10 cells 76
showing bright fluorescence were selected from one view field, in which more than 100 77
cells existed, and the fluorescent-signal intensity of each cell and background signal 78
intensity were measured using the image processing program ImageJ. Perceval HR 79
fluorescent intensity ratio of a cell was obtained through the following formula, 80
[{(fluorescent intensity of a UV/violet-light excited cell)-(background fluorescent 81
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intensity of the UV/violet-light excited field)}/ {(fluorescent intensity of the blue-light 82
excited cell)-(background fluorescent intensity of the blue-light excited field)}]. More 83
than 50 cells were analyzed per condition, and their data distribution is expressed as a 84
standard box-and-whisker plot in Figure 2(a). 85
Biochemical assay for cellular ADP/ATP ratio: After harvest, cells were quickly lysed 86
in 80°C hot buffered ethanol (HEPES 10 mM, 75% ethanol, pH 7.1) with occasional top-87
speed vortexing (15 sec each with 30 sec interval; 3 times; Ref. [9]). The resulting cell 88
extracts were then dried up by evaporation and checked for ADP/ATP ratio using 89
EnzyLight ADP/ATP Ratio Assay Kit (BioAssay Systems), by which ATP amount is 90
measured by D-luciferin and firefly luciferase-based luminescence, while ADP amount 91
is measured through its enzymatic conversion to ATP. 92
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Results and Discussion 94
Probably because of high expression of PercevalHR, cells carrying pRS425-95
PercevalHR grew slightly slowly (Figure 1(a); compare the pRS425-Perceval cells 96
(without antimycin A, gray closed square) to the pRS425 cells (without antimycin A, 97
black closed diamond). It should be noted that the PercevalHR fluorescent signal was 98
sometimes hardly detected quantitatively under our experimental conditions described 99
below when it was expressed at lower levels. We think that the growth retardation 100
approximately 12-hr after culture start represents diauxic shift, because, after this time 101
point, cells accumulated reactive oxygen species, which are byproducts of mitochondrial 102
respiration (data not shown). 103
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We then observed cells carrying pRS425-PercevalHR as described in the Materials 104
and Method section. According to the fluorescent images shown in Figure 1(b), 105
PercevalHR was diffusively and uniformly distributed in the cells with dark circles 106
representing vacuoles (compare the fluorescent images with the light-field images). As 107
expected, cells transformed with the empty vector pRS425 did not exhibit any 108
fluorescence (Figure 1(b)). 109
We next quantitated the fluorescence intensity of cells carrying pRS425-PercevalHR 110
as described in the Materials and Methods section. The ratios of the green fluorescence 111
intensity of UV/violet light-illuminated cells against that of blue light-illuminated cells 112
were significantly different between pre and post diauxic shift (Figure 2(a); compare box 113
3 with boxes 1 and 2). The shorter-wavelength shift in the PercevalHR-excitation light 114
along diauxic shift is consistent with our expectation, since, as aforementioned, 115
mitochondrial respiration is known to produce ATP slower than fermentation. 116
When cells were cultured in the presence of the mitochondrial inhibitor antimycin A, 117
the shorter shift in the excitation wavelength was more drastic in the case of the 15-hr-118
cultured post-diauxic-shift samples (Figure 2(a), compare box 6 with 3). Such an effect 119
of antimycin A was also observed in the case of 9-hr-cultured late log-phase samples 120
(Figure 2(a), compare box 5 with 2) but not in the case of 3-hr-cultured early log-phase 121
samples (Figure 2(a), compare box 4 with 1). This result may reflect a contribution of 122
mitochondrial respiration for ATP production in late log phase, and is consistent with 123
slight growth retardation by antimycin A at this time point (Figure 1(a), compare pRS425-124
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PercevalHR cells cultured with antimycin A (open circle) to those cultured without 125
antimycin A (gray closed square)). 126
In the experiment shown in Figure 2(b), cells were cultured as done Figures 1(a) and 127
2(a) and biochemically checked for the cellular ADP/ATP ratio as shown in the Materials 128
and Methods section. We then noticed that cellular expression of PercevalHR did not 129
considerably affect the cellular ADP/ATP ratio (Figure 2(b), see the leftmost two 130
columns). Moreover, it should be noted that changing pattern upon long-time culturing 131
and/or antimycin A treatment shown in Figure 2(b) did not largely differ from that shown 132
in Figure 2(a). This result indicates that PercevalHR actually acts as a probe for the 133
cellular ADP/ATP ratio. 134
Perceval and Perceval HR have been reported to change their excitation spectra not 135
only depending on the ADP/ATP ratio, but also by the surrounding pH condition (shorter-136
wavelength shift by acidic conditions). In order to confirm that the changes in the 137
fluorescence-intensity ratio shown in Figure 2(a) are not because of intracellular pH, we 138
monitored it using another cytosolic fluorescent reporter protein Rosella, which is a fusion 139
of a pH-sensitive GFP variant (low fluorescence under low pH condition) and a pH-140
resistant red fluorescent protein. As shown in Figure 3, cells transformed with the Rosella-141
expression plasmid pAS1NB c Rosella I [10] were analyzed by dual-color flowcytometry 142
using the BD Accuri C6 flowcytometer. Consistent with a previous report showing that 143
severe nutrient depletion lowers cytosolic pH in S. cerevisiae cells [11], suspension of 144
Rosella-expressing cells in pure water seemed to decrease the green fluorescence of 145
Rosella (Figure 3, lower position of the plots in panel (c) in comparison with the other 146
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panels). On the other hand, cellular treatment with antimycin A or diauxic shift did not 147
seem to change the fluorescent property of Rosella or did slightly increased its green 148
fluorescence (see the merged data shown in panels (f) to (i)). We thus think that, at least 149
under the experimental conditions employed in this study, diauxic shift and/or antimycin 150
A treatment did not result in acidification of the cytosol. 151
In conclusion, by using the fluorescent reporter protein PercevalHR, we successfully 152
demonstrated increment of the intracellular ADP/ATP ratio upon diauxic shift, which is 153
enhanced by the mitochondrial inhibitor antimycin A, in yeast cells. We anticipate that 154
PercevalHR and its expression plasmid pRS425-PercevalHR are valuable tools for 155
physiological and pathological studies using yeast cells as a model organism. This 156
technique can be an alternative to the biochemical assays, which is accompanied with cell 157
disruption and may cause an unexpected experimental error, for example, caused by ATP 158
hydrolysis during cell lysis. 159
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Acknowledgement 161
We deeply thank to Prof. Kenji Kohno and Prof. Hiroshi Takagi (Nara Inst. Sci. Tech.) 162
for a wide variety of valuable supports, which have been indispensable for this study. The 163
original PercevalHR plasmid, GW1-PercevalHR, was a gift from Dr. Gary Yellen 164
(Addgene plasmid # 49082). pAS1NB c Rosella I was a gift from Dr. Mark Prescott 165
(Addgene plasmid # 71245). 166
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Author Contribution 168
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PTMN and YIK constructed DNA plasmids and performed the experiments. YK designed 169
the experiments and completed the manuscript. 170
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Disclosure Statement 172
No potential conflict of interest was reported by the authors. 173
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Funding 175
This research was supported by Ohsumi Frontier Science Foundation. 176
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References 178
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shift and stationary phase. OMICS. 2010; 14: 629-638. 187
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[8] Mai CT, Le QG, Ishiwata-Kimata Y, et al. 4-Phenylbutyrate suppresses the unfolded 195
protein response without restoring protein folding in Saccharomyces cerevisiae. FEMS 196
Yeast Res. 2018; 18: foy016. 197
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[10] Rosado CJ, Mijaljica D, Hatzinisiriou I, et al. Rosella: a fluorescent pH-biosensor 200
for reporting vacuolar turnover of cytosol and organelles in yeast. Autophagy. 2008; 4: 201
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[11] Orij R, Postmus J, Ter Beek A, et al. In vivo measurement of cytosolic and 203
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278. 206
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Figure Legends 207
Figure 1. Expression of PercevalHR in S. cerevisiae cells 208
(a) KMY1005 cells transformed with pRS425-PercevalHR or pRS425 were grown in 209
liquid SD medium, and optical density of the culture was checked as described in the 210
Materials and Methods section. Antimycin A (final concentration of 20 µg/ml) was added 211
into the medium upon culture start. (b) KMY1005 cells transformed with pRS425-212
PercevalHR or pRS425 were cultured without antimycin A as done in panel A, and 213
observed under the fluorescence microscope. 214
215
Figure 2. Change in ADP/ATP ratio in S.cerevisiae cells 216
(a) Fluorescent images were obtained and quantitated as described in the Materials and 217
Methods section. *** p< 0.001. (b) KMY1005 cells carrying pRS425 or pRS425-218
PercevalHR were subjected to biochemical analysis for cellular ADP/ATP ratio as 219
described in the Materials and Methods section. Antimycin A (final concentration of 20 220
µg/ml) was added into the medium upon culture start. 221
222
Figure 3. Change in the fluorescence property of Rosella in S. cerevisiae cells 223
KMY1005 cells transformed with pAS1NB c Rosella I were cultured as per Figure 1 or 224
suspended in pure water for 10 min before flowcytometric analysis (threshold setting: 225
FCS 8,000 and FL2 500). The time point of diauxic shift was determined according to 226
cellular growth and ROS production (data not shown). Dead cells with high 227
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autofluorescence (the FL4 values of more than 900) are not shown in the plots. Antimycin 228
A (final concentration of 20 µg/ml) was added into the medium upon culture start. 229
230
Figure1 (a)
3 hr
-cul
ture
d ce
lls(F
erm
enta
tively
grow
ing)
(b)
Cul
ture
opt
ical
den
sity
(OD
)60
0
Time after culture start (hr)
UV/violet-light excitation/Greenfluorescence
Blue-light excitation/Green fluorescence Light field
0.25
0.5
1
2
4
0 2 4 6 8 10 12 14 16 18 20
15 h
r-cul
tued
cells
(Pos
t-dia
uxic
shi
ft)pRS425
pRS425-PercevalHR
pRS425-PercevalHR(+ antimycin A)
3 hr
-cul
ture
d ce
lls(F
erm
enta
tively
grow
ing)
15 h
r-cul
tued
cells
(Pos
t-dia
uxic
shi
ft)pRS
425
p
RS
425-
Per
ceva
lHR
Figure2
******
******
******
1 2 3 4 5 6
Time after culture start (hr)
3 9 15 3 9 15
Non-treat + Antimycin A
Per
ceva
l HR
fluo
resc
ent i
nten
sity
ratio
(UV
/vio
let-l
ight
exc
itatio
n / b
lue-
light
exc
itatio
n)
(a)
(b)
0
0.2
0.4
0.6
0.8
3 3 9 15 3 9 15
Plasmid pRS425-PercevalHR
pRS
425
– +Antimycin A
Time after culture start (hr)
Bio
chem
ical
ly a
ssay
ed A
DP
/ATP
ratio
400
4000
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400000
4000000
100 1000 10000 100000
Figure3
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FL3(red)FL3(red) FL3(red)
FL3(red)FL3(red) FL3(red)
FL1(green
)FL1(green
)
FL1(green
)
Cells poorly expressing Rosella
Cells poorly expressing Rosella
Cells poorly expressing Rosella
Cells poorly expressing Rosella
Cells poorly expressing Rosella
(a) 5 hr BEFORE diauxic shift (b) 5 hr BEFORE diauxic shift (c) 30 min in pure water
+ antimycin A
(d) 5 hr AFTER diauxic shift (e) 5 hr AFTER diauxic shift
+ antimycin A
(f) Merge (panel A (yellow colored)and panel B (blue colored)
400
4000
40000
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100 1000 10000 100000
(g) Merge (panel A (yellow colored)and panel D (blue colored)
100 1000 10000 100000
(h) Merge (panel D (yellow colored)and panel E (blue colored))
100 1000 10000 100000
(i) Merge (panel B (yellow colored)and panel E (blue colored))
100 1000 10000 100000
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Graphical abstract caption 231
Yeast cells producing PercevalHR emit green fluorescence, excitation spectrum of which 232
varies dependently on cellular ATP/ATP ratio. 233
Yeast cells producing PercevalHR from pRS425-PercevalHR
・Rapid growth by fermentation(Low ADP)
UV/violet-light excitation
Blue-light excitation
・Respiration・Mitochondria dysfunction
(High ADP)
UV/violet-light excitation
Blue-light excitation