DEPARTMENT OF BIOLOGICAL AND ENVIRONMENTAL SCIENCES

Study the Toxicity Mechanism of Gallium

and Indium in Yeast Saccharomyces

cerevisiae

Liqian Zhao

Degree project (45 hec) for Master of Science (120 hec) in Ecotoxicology [2019], Second Cycle

Supervisor: Markus Tamas, Department of Chemistry & Molecular Biology, University of Gothenburg

Examiner: Jonas Warringer, Department of Chemistry & Molecular Biology, University of Gothenburg

Abstract

Gallium and indium are widely used in electronics and energy industry in recent years.

They are non-essential elements for living organisms and pose risks to both the

environment and human health. Previous high throughput screening in our lab

identified single deletion mutants that were sensitive to aluminum, gallium and

indium in yeast Saccharomyces cerevisiae (S. cerevisiae). In this study, we tested the

sensitivity of 32 mutants from the screening in the growth assay, the deleted genes of

which were mainly involved in metal homeostasis and unfolded protein response

(UPR). We then tried to identify the toxicity mechanism at the molecular level.

Although deletion mutants with genes involved in the UPR pathway displayed

hypersensitivity to gallium, we did not detect UPR activation under gallium stress in a

short time. Results were verified by checking Ire1 (UPR sensor protein) clustering,

HAC1 (transcription factor activated by ER stress) mRNA splicing and quantitative

PCR of UPR downstream genes. Through monitoring the nuclear localization of

different fluorescence fused transcription factors (GFP-Crz1 and Pho4-GFP) and

DNA damage repair proteins (Rad52-YFP and Rfa1-YFP), we observed that the

percentage of cells with GFP-Crz1 nuclear foci was significantly higher under

aluminum and indium stress, which suggested aluminum and indium may influence

calcium homeostasis. However, the connection of aluminum, gallium and indium

stress with DNA damage and phosphate homeostasis was not found. Our findings

provide valuable suggestions for future research of gallium and indium toxicity

mechanisms in yeast.

Table of contents

List of abbreviations .................................................................................................... 1

Introduction .................................................................................................................. 2

Properties and usage of gallium and indium .............................................................. 2

Environmental concentrations of gallium and indium ............................................... 3

Gallium and indium toxicity to human ....................................................................... 4

Ecotoxicological information of gallium and indium ................................................ 5

General mechanism of metal toxicity ......................................................................... 5

The toxicity mechanism of gallium and indium ......................................................... 6

ER stress and unfolded protein response (UPR) ........................................................ 7

Research aim .............................................................................................................. 8

Materials and Methods ................................................................................................ 8

Chemicals ................................................................................................................... 8

Strains, plasmids and primers ..................................................................................... 8

Strains .................................................................................................................. 8

Plasmids .............................................................................................................. 9

PCR primers ...................................................................................................... 10

Yeast medium and culturing conditions ................................................................... 10

Yeast transformation (short protocol) ....................................................................... 11

Growth assay on solid medium ................................................................................ 11

RNA analysis ............................................................................................................ 11

RNA extraction, cDNA synthesis and PCR assay .............................................. 11

Quantitative reverse transcription PCR (RT-qPCR) ......................................... 12

Fluorescence microscopy ......................................................................................... 13

Results ......................................................................................................................... 13

Several yeast mutants were sensitive to Al3+

, Ga3+

and In3+

.................................... 13

UPR was unlikely induced by Ga3+

stress ................................................................ 15

Ire1 was not clustered in yeast cells exposed to Ga3+

....................................... 16

HAC1 mRNA was not spliced under Ga3+

stress ............................................... 16

UPR-target genes were not up-regulated by a short time of Ga3+

stress .......... 17

Measurement of other physiological changes under Al3+

, Ga3+

and In3+

stress ....... 20

DNA damage ..................................................................................................... 20

Al3+

and In3+

affected calcium homeostasis ...................................................... 21

Phosphate homeostasis under metal stress ....................................................... 22

Discussion.................................................................................................................... 23

Conclusion .................................................................................................................. 25

Acknowledgment ........................................................................................................ 25

References ................................................................................................................... 25

1

List of abbreviations

GFP: Green fluorescent protein

YFP: Yellow fluorescent protein

PCR: Polymerase chain reaction

RT-qPCR: Quantitative reverse transcription PCR

WT: Wild type

SC-HIS: Synthetic dropout medium without histidine

SC-URA: Synthetic dropout medium without uracil

DTT: Dithiothreitol

ITO: Indium tin oxide

GaAs: Gallium arsenide

GaN: Gallium nitride

LED: Light-emitting diode

OD600: Optical density at 600nm

ssDNA: Single strand DNA

ER: Endoplasmic reticulum

UPR: Unfolded protein response

pg/m3: Picograms per cubic meter

EC50: Half maximal effective concentration

LC50: Lethal concentration resulting in 50 percent mortality

EC10: 10% effect concentration based on the concentration-response relationship.

NOEC: No observed effect concentration

PNEC: Predicted no effect concentration

KD: Concentration of a chemical in soil/concentration of the substance in water

ROS: Reactive oxygen species

U.S.: The United States

ECHA: The European Chemicals Agency

REACH: European regulation on Registration, Evaluation, Authorisation, and

Restriction of Chemicals

2

Introduction

Properties and usage of gallium and indium

Gallium and indium are soft, silvery-white metals in the same group of the periodic

table, and share similar chemical properties. Free Ga3+

exists at pH less than 3, and

Ga(OH)4− mainly exists at pH values higher than 4.5. At pH values below 4.5, indium

exists as free In3+

, and at neutral pH, it forms In (OH)30. Both gallium and indium

tend to complex with hard ligands such as OH−, F

−, and acetate. They are relatively

insoluble and tend to interact with hydroxide in the aqueous environment (Wood and

Samson 2006)

As GaAs or GaN, gallium is applied in integrated circuits and optoelectronic

devices such as laser diodes, LEDs, and solar cells. Pure gallium (Ga0) is used to

make high-temperature thermometers and brilliant mirrors. Due to the unique

properties of thermal convection, liquid gallium (Ga0) is used to study planetary and

astrophysical magnetohydrodynamics. Gallium (Ga3+

) is also used as

chemotherapeutic medicine in cancer treatment (Jakupec and Keppler 2004;

Chitambar 2010). Pure indium (In0) is used in vapor deposition, plating applications,

and soldering. Indium oxide doped with tin which forms indium tin oxide (ITO) is

used as an integral material in flat panel displays, LEDs to transform electrical data

into an optical form, and is also applied in photovoltaic cells. The phosphide form of

indium (InP) is used in semiconductors. Radioisotope 111

In is applied in medical

imaging and biodistribution studies (Foley et al. 2017; Shanks et al. 2017).

In the past 40 years, the production and usage of gallium and indium increased

dramatically due to the development of the electronic industry and energy

technologies. Data from the National Minerals Information Center of the U.S.

Geological Survey showed that the global production of gallium increased more than

20 times (from 16 t to 469 t), and indium increased more than ten times (from 68.4 t

to 769 t) since the 1970s (Figure 1). Correspondingly, the end-use of gallium in the

U.S. rose up to 5 times and indium usage increased about 30 times in 28 years (Figure

2) (Kelly et al. 2016).

Figure 1. World production of gallium and indium (Kelly et al. 2016).

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Figure 2. End-use of gallium and indium in the U.S. (Kelly et al. 2016).

Environmental concentrations of gallium and indium

Natural gallium and indium elements are present mostly in aluminum and zinc ores

and in coals. As a result of human activities such as mining and ore processing, coal

combustion, improper electronic waste disposal and accidental spill, their

environmental concentrations in air, water, and living organisms increased in recent

decades. The behavior of gallium and indium in the environment were reported in a

few studies. The distribution coefficient (KD) values of gallium and indium were

measured to be 2021 and 408 L/kg respectively at pH 5.95, which decreased with

higher pH. The KD reflects gallium and indium have a strong bind with soil. The

mobility of gallium and indium is low at neutral pH but increases with environmental

acidity. Therefore, soil or dust ingestion may be the most likely exposure pathway for

humans and animals (Jensen, Gaw et al. 2018).

The concentrations of gallium and indium in groundwater were monitored in

Taiwan, which ranged at 7.91–71.39 μg/L and 0.95–20.05 μg/L respectively in the

industry park (Chen 2006). The average levels of gallium and indium in the

semiconductor industrial science park were 19.34 μg/L and 9.25 μg/L respectively,

which were significantly higher than the reference sites (0.01–0.05 μg/L) (Chen 2006).

The concentrations of dissolved and particulate indium of 50 samples from rivers,

lakes and coastal seawaters were measured in Japan, which ranged 1.4–3.0 and

2.4–9.1 ng/L respectively (Miyazaki, Kimura et al. 2012). It was found samples from

sites near big chemical factories, thermoelectric power plants, petroleum refining

plants, and highways had much higher concentrations of indium. The indium

concentration of a headwater stream contaminated by acid mine drainage in

southwestern Colorado was reported to be 6–29 μg/L, which was 10,000 times of

those found in neutral rivers (White, Hussain et al. 2017).

Using a spectrophotometric method, the gallium contents in different soils were

measured in Poland, which ranged from 41.7 to 437 μg/g. Also, an increased gallium

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Research and development

Other

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Coatings

Solders and alloys

Electrical components and semiconductors

Research and other uses

4

content (around 300–400 μg/g) was found in the soils from an industrial area close to

a zinc plant and a nonferrous metal plant. Moreover, higher gallium concentration

(218 μg/g) was detected in one agriculture soil sample, while the lowest concentration

was around 40 μg/g (Połedniok, Kita et al. 2012). Indium concentrations in the air

were shown to be around 0.05 to 20 pg/m3 in remote regions (South Pole, Shetland

Islands), however, the concentration near an industrial area in Japan went up to 1200

pg/m3 (Shanks et al. 2017). Since gallium and indium are often doped with other

metals in high-performance alloys, recycling nowadays is still a challenging problem.

Gallium and indium toxicity to human

Both gallium and indium are non-essential elements for living organisms and do not

play any known physiological role in biochemical functions. Workers in the

semiconductor industry and mine plants are highly affected by gallium and

indium-associated particles. In a previous study, the examination of 103

optoelectronic workers and 67 control individuals in Taiwan showed the plasma

levels of malondialdehyde (MDA, a byproduct of lipid peroxidation processes) in

gallium exposed workers were significantly higher, indicating increased lipid

peroxidation by gallium stress (Liao, Hwang et al. 2006). The malondialdehyde level

was correlated significantly with the urinary gallium level. The acute effects of

accidental gallium fume exposure in the laboratory were reported before, including

increased rash and neurological pain, heart rate, shortness of breath, vertigo and

headaches (Meigs 1972; Ivanoff, Ivanoff et al. 2012). Gallium was shown to induce

the death of tumor cells (Chitambar 2010), the potential risks to healthy cells were

therefore deduced from the same mechanism such as perturbing cellular iron

metabolism.

The examination of 87 current Indium tin oxide (ITO) workers revealed there was

a correlation between elevated indium exposures and health problems. It was shown

high indium exposure through inhalation would induce dyspnea, lower spirometric

parameters, and higher levels of serum lung disease biomarkers (Cummings, Virji et

al. 2016). The indium concentrations in urine and plasma of nine workers and 5

retired workers from a metallurgical department in Belgium were monitored (Hoet,

De Graef et al. 2012), and results showed the highest indium concentration

(0.32–12.61 μg/L in plasma; 0.22–3.50 μg/g in urine) in the current workers,

compared with the retired workers (0.03–4.38 μg/L in plasma; 0.02–0.69 μg/g in urine)

and the control group (< 0.03 μg/L in plasma; < 0.02 μg/g in urine). These studies

suggested the insoluble indium compounds could be absorbed and accumulate in

human body, which could lead to "endogenous exposure".

Our knowledge about the toxicity of gallium and indium in human is still limited.

Since they are in the same group with aluminum, probably they share similar toxic

effects such as damage to the nervous system, as well as impaired performance in

motor, sensory, and cognitive function. Moreover, the increased usage and

environmental accumulation demand a better understanding of indium and gallium

toxicity mechanisms.

5

Ecotoxicological information of gallium and indium

The toxicity of gallium and indium to the ecosystem, which was determined by the

EC50/LC50 (respecting the short-term toxicity) and NOEC/EC10 (indicating the

long-term toxicity) of organisms on different trophic levels, was discussed in several

studies. These values were divided by an assessment factor to get the PNEC

(predicted no effect concentration) for chemical risk assessment. The toxicity data for

indium (In3+

) is available from the European Chemicals Agency (ECHA), where they

collect 12 LC/EC50s values of 9 different species, 11 NOEC/EC10 values of 7

different species from 3 trophic levels (algae, invertebrates and fish) and 4 NOEC

values of terrestrial organisms (reference see REACH). In aquatic system the

EC/LC50s ranged from 1 to more than 400 mg/L and NOEC/EC10s from 92 to

25,000 μg/L. For terrestrial organisms, the NOECs were observed at around 0.032 to

2.23 g In/kg.

The ecotoxicological data for gallium was not found from the ECHA database,

and there was less information available in the literature. The LC50 of carp (Cyprinus

carpio Linnaeus) under the exposure of gallium for 96 h, was reported to be

95.6 ± 14.3 mg/L (Betoulle, Etienne et al. 2002). The acute toxicity of gallium in

brackish and marine organisms (Americamysis bahia, Brachionus plicatilis and

Artemia salina) was measured, and the LC50 values ranged from 10 to 50 mg/L

(Onikura, Nakamura et al. 2005). The IC50 (concentration that inhibited 50% of the

population) of gallium on the fertilization of coral (Acropora tenuis) eggs was 3.4

mg/L (Negri, Harford et al. 2011). However, gallium and indium levels are usually

not measured in the mine drainage water and not under any regulation, except limit

restrictions for specific indium exposed workers. In the future, these toxicity

endpoints will make more sense if the metal concentrations are monitored in the

environment.

General mechanism of metal toxicity

The mechanism of metal toxicity was widely studied with heavy metals such as

arsenic, cadmium, chromium, lead, and mercury. Some modes of action were

relatively clear, for instance, how the metals enter and efflux the cell and how they

affect the cellular functions such as protein folding and nutrient metabolism. The

revealed mechanism in microorganisms and plants was discussed thoroughly in recent

reviews (Wysocki and Tamas 2010; Lemire, Harrison et al. 2013; Kupper and

Andresen 2016), which provides a basic understanding of the toxicity to animals and

human.

Firstly, toxic metals induce intracellular reactive oxygen species (ROS)

production and antioxidant depletion. This metal-induced oxidative stress is probably

due to the Fenton reaction (Fe2+

+ H2O2 → Fe3+

+ OH– + OH

•, Fe

3+ + O2

•– → Fe

2+ +

O2), either through the catalysis activity of toxic metals (such as Cu2+

, Cr3+

and Co2+

),

or through Fenton-active Fe in the cytoplasm released through competitively binding

of toxic metal (such as Ag1+

, Hg2+

and Ga3+

) to the cellular Fe ligands. Some metals

such as Ag1+

, Cd2+

, Co2+

and Zn2+

form covalent bonds with sulfur leading to the

oxidation of cellular thiols, which in turn causes unwanted protein disulphide and

6

destroys the function of some antioxidants such as glutathione. Secondly, toxic metals

cause protein dysfunction and loss of enzyme activity. The metal-induced ROS lead

to the oxidation of protein amino acids, which inactivates the protein and results in

degradation. Additionally, the replacement by other metals at specific metal-binding

sites also inhibits the activity of enzymes. Thirdly, toxic metals may impair membrane

function. Excess of toxic metals was shown to disturb the integrity of the plasma

membrane, probably due to lipid peroxidation. Besides, toxic metals were shown to

influence nutrient assimilation by, for example, the competitive inhibition of sulphate

uptake or interfering with Fe uptake. Finally, the genotoxicity of toxic metals was

demonstrated by many studies. Metal induced Fenton-action also counts for DNA

damage. (Lemire, Harrison et al. 2013)

In addition to the common mechanisms mentioned above, some heavy metals

have specific toxic effects. For instance, lead could impair the neuron system through

perturbing the calcium homeostasis (Tchounwou, Yedjou et al. 2012), and arsenic or

cadmium could induce protein misfolding and aggregation (Tamás, Sharma et al.

2014).

The toxicity mechanism of gallium and indium

Relatively, there was less information about the toxicity mechanisms of gallium and

indium than the well-studied heavy metals. With the expansion of the electronic

industry and increased usage of these two metals, more research about the toxicity

mechanisms was conducted in microorganisms, plants, fishes and cell lines in recent

years. As far as we learned, gallium and indium may disturb metal homeostasis

especially iron (Kaneko, Thoendel et al. 2007), as well as induce endoplasmic

reticulum (ER) stress and increase ROS production (Brun, Christen et al. 2014).

Through genome-wide phenotypic screening, a set of aluminum (Al3+

) tolerant

mutants were identified in yeast S. cerevisiae, six of which (cka2Δ, ipt1Δ, lem3Δ,

ppm1Δ, ume1Δ and YLR407WΔ) also showed hypertolerance to dissolved gallium

(Ga3+

) (Tun, O'Doherty et al. 2014). The genes were related to different cell functions:

CKA2 encodes a subunit of casein kinase 2, IPT1 encodes inositolphosphotransferase,

LEM3 is an endocytosis-related gene, PPM1 is a protein phosphatase regulator and

UME1 is a component of histone deacetylase complexes. It was reported gallium

could be taken in by the bacteria Pseudomonas aeruginosa and inhibited its growth,

due to the repression of iron (Fe) uptake in a non-competitive way (Kaneko, Thoendel

et al. 2007). The microarray analysis in this study showed 27 genes were activated

and 47 genes repressed by Ga3+

stress. These genes are involved in carbon utilization,

ribosome, and Fe homeostasis. Additionally, the transcriptional regulator pvdS and

respective downstream genes which are induced by Fe starvation were also

down-regulated during Ga3+

exposure (Kaneko, Thoendel et al. 2007).

A study on plant Arabidopsis thaliana showed gallium could increase the

production of malondialdehyde (MDA), and up-regulate the expression of the

transporters AtALMT and AtMATE which are responsible for citrate and malate

secretion (Chang, Wang et al. 2017). Similar effects were also found for aluminum in

this study. A comparative DNA microarray analysis of gallium-resistant and

gallium-sensitive lymphoma cell lines showed elevated gene expression of the

7

metal-responsive transcription factor metallothionein-2A, and the zinc transporter

ZnT-1 in gallium-resistant cells (Yang, Kroft et al. 2007). The mRNA level of

metallothionein-2A was further confirmed by semiquantitative PCR and Northern blot

analysis. Consistently, Western blot showed a higher expression level of

metallothionein-2A protein. It was speculated in this study that zinc could induce the

expression of metallothionein to protect cells against the gallium toxicity.

The toxicity mechanism of indium nitrate (In(NO3)3) and ITO nanoparticles was

investigated in liver cells of zebrafish (Danio rerio), which showed exposure of

In(NO3)3 for 24 h would trigger ER stress response and induce ROS production (Brun,

Christen et al. 2014). A significant activation of ER stress related genes (bip, atf6,

chop and synv) was detected. The elevated ROS production was accompanied by the

increased concentration of In(NO3)3. The mRNA levels of cat and mu1 which are

indicators of oxidative stress were also higher under indium stress. In addition,

expression levels of apoptosis-related genes bcl-2, p53, noxa, apaf1, casp9, and

inflammatory response genes tnf-a, irf3, irf9, nfkb1/p50 changed significantly. Similar

results were observed in zebrafish embryos. Besides, they noticed most of the effects

were much weaker when cells were exposed to ITO.

ER stress and unfolded protein response (UPR)

Most membrane proteins and secreted proteins are folded and get mature (through

post-translational modification to form a functional protein) in the ER. Some stresses

such as heavy metals and chemicals (tunicamycin or DTT) that affect protein disulfide

bond formation, will cause the accumulation of misfolded proteins in the ER. Then

the UPR will be induced and a set of genes will be activated to help protein folding

and alleviate the ER stress. There are three typical UPR pathways, which are defined

by the type of the transmembrane ER-sensor protein: Ire1 (inositol requiring

enzyme-1) in lower eukaryotes, PERK (double-stranded RNA-activated protein

kinase like ER kinase) and ATF6 (activating transcription factor 6) in metazoan

organisms (Walter and Ron 2011).

The Ire1 pathway conserved in all eukaryotes is responsible to cope with ER

stress in yeast (Figure 3). When ER stress is induced, Ire1 which is a transmembrane

sensor protein undergoes trans-autophosphorylation and oligomerization to get

activated. Then HAC1 mRNA is spliced by the RNase domain of Ire1, thus the

transcription factor Hac1 is synthesized and enters the nucleus to regulate UPR target

genes (Wu, Ng et al. 2014). UPR targets are genes encoding ER chaperones,

translocation proteins and protein disulfide bond formation regulators, or assisting

proteins glycosylation, modification and degradation (Kimata, Ishiwata-Kimata et al.

2006). Meanwhile, the expression of transmembrane and secreted proteins is

repressed to reduce proteins that enter ER. The principle of the regulation is to restrict

protein loading in the ER, promote protein folding by ER chaperones, trigger

ER-associated protein degradation (ERAD) or induce cellular apoptosis if the

alleviating response fails (Travers, Patil et al. 2000).

8

Figure 3. UPR pathway in yeast (Wu, Ng et al. 2014).

Research aim

The aim of this study was to confirm the sensitivity to aluminum, gallium and indium,

in the yeast S. cerevisiae mutants which were identified from previous high

throughput screening in our lab (Schulze 2018). Furthermore, try to illustrate possible

mechanisms of how the involved genes are related to indium and gallium toxicity: if

the metals induce UPR, cause DNA damage or affect Ca2+

and phosphate

homeostasis.

Materials and Methods

Chemicals

Peptone from meat, granulated (Merck KGaA, cat. no. 91249),

Yeast extract granulated, (Merck KGaA, cat. no. 103753)

Yeast Nitrogen Base (YNB) without ammonium sulfate and amino acids, powder

(Formedium Ltd., cat. no. CYN0502)

Yeast Nitrogen Base (YNB) without phosphate and amino acids, powder (Formedium

Ltd., cat. no. CYN0801)

Complete CSM mixture and CSM mixture of different drop out (Formedium Ltd.)

Ammonium sulphate, powder (Merck KGaA, cat. no. 1012171000)

D-Glucose (VWR International, cat. no. BDH9230-2.5KG)

Aluminum sulfate, Al2(SO4)3(H2O)18, purity 99.4%, (BDH Laboratory)

Indium chloride, InCl3, powder, purity 98%, (Sigma-Aldrich, cat. no. 334065-10g)

Gallium chloride, GaCl3, granulated, purity 5-nines (99.999%), (Indium Corporation)

Strains, plasmids and primers

Strains

Yeast strains used in this study are listed below in Table 1.

9

Table 1. Yeast strains used in this study

Strains Genotype Source/Reference

BY4741 (WT) MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 EUROSCARF

BY4741 der1Δ BY4741 der1Δ::kanMX EUROSCARF

BY4741 hac1Δ BY4741 hac1Δ::kanMX EUROSCARF

BY4741 ire1Δ BY4741 ire1Δ::kanMX EUROSCARF

BY4741 ubp3Δ BY4741 ubp3Δ::kanMX EUROSCARF

BY4741 ubp14Δ BY4741 ubp14Δ::kanMX EUROSCARF

BY4741 ptc3Δ BY4741 ptc3Δ::kanMX EUROSCARF

BY4741 ubx6Δ BY4741 ubx6Δ::kanMX EUROSCARF

BY4741 mpd2Δ BY4741 mpd2Δ::kanMX EUROSCARF

BY4741 ecm1Δ BY4741 ecm1Δ::kanMX EUROSCARF

BY4741 ecm5Δ BY4741 ecm5Δ::kanMX EUROSCARF

BY4741 ecm6Δ BY4741 ecm6Δ::kanMX EUROSCARF

BY4741 arr3Δ BY4741 arr3Δ::kanMX EUROSCARF

BY4741 arr1Δ BY4741 arr1Δ::kanMX EUROSCARF

BY4741 cnb1Δ BY4741 cnb1Δ::kanMX EUROSCARF

BY4741 cch1Δ BY4741 cch1Δ::kanMX EUROSCARF

BY4741 pmr1Δ BY4741 pmr1Δ::kanMX EUROSCARF

BY4741 pho3Δ BY4741 pho3Δ::kanMX EUROSCARF

BY4741 pho5Δ BY4741 pho5Δ::kanMX EUROSCARF

BY4741 ldh1Δ BY4741 ldh1Δ::kanMX EUROSCARF

BY4741 slc1Δ BY4741 slc1Δ::kanMX EUROSCARF

BY4741 sct1Δ BY4741 sct1Δ::kanMX EUROSCARF

BY4741 sur1Δ BY4741 sur1Δ::kanMX EUROSCARF

BY4741 csg2Δ BY4741 csg2Δ::kanMX EUROSCARF

BY4741 cst26Δ BY4741 cst26Δ::kanMX EUROSCARF

BY4741 scs22Δ BY4741 scs22Δ::kanMX EUROSCARF

BY4741 pso2Δ BY4741 pso2Δ::kanMX EUROSCARF

BY4741 rad5Δ BY4741 rad5Δ::kanMX EUROSCARF

BY4741 rad14Δ BY4741 rad14Δ::kanMX EUROSCARF

BY4741 psy3Δ BY4741 psy3Δ::kanMX EUROSCARF

BY4741 ogg1Δ BY4741 ogg1Δ::kanMX EUROSCARF

BY4741 ung1Δ BY4741 ung1Δ::kanMX EUROSCARF

BY4741 rdh54Δ BY4741 rdh54Δ::kanMX EUROSCARF

BY4741 slt2Δ BY4741 slt2Δ::kanMX EUROSCARF

W303 with

Ire1-GFP

W303- ire1Δ (MATa leu2-3,112 trp1-1 can1-100

ura3-1 ADE2 his3-11,15, ire1Δ::TRP1)/ Ire1-GFP

(Le, Ishiwata-Kimata et al.

2016)

W3749-14C W303-1A, ade2 bar1::LEU2 RAD52-YFP (Litwin, Bocer et al. 2013)

W3775-12C W303-1A, ade2 bar1::LEU2 RFA1-YFP (Litwin, Bocer et al. 2013)

Plasmids

Plasmids used in this study are listed below in Table 2.

10

Table 2. Plasmids used in this study

Plasmid Description Reference

pRS313-IRE1 IRE1 with its own promoter inserted in pRS313 (Kimata, Oikawa et al. 2004)

Ire1-GFP pRS313-TRF1p-IRE1-GFP (Le, Ishiwata-Kimata et al. 2016)

pA1 ACR1-ACR2-ACR3 (4.2kb insert) in BamHI site

of pFL44L

(Bobrowicz, Wysocki et al.

1997)

GFP-CRZ1 GFP-tagged CRZ1 in pUG36 (Stathopoulos-Gerontides, Guo

et al. 1999)

Pho4-GFP pRS416 with Ppho4-Pho4-GFP (Desfougeres, Gerasimaite et al.

2016)

PCR primers

PCR primers used in this study are listed below in table 3.

Table 3. Primers used in this study

Name Sequence(5’-3’) Use

HAC1-a AGGAAAAGGAACAGCGAAGG Detection of HAC1 mRNA

splicing(Gardarin, Chedin et al. 2010) HAC1-b GAATTCAAACCTGACTGCGC

KAR2-2F TTTTAGTTAGAGGTGCCGAT RT-qPCR for UPR downstream gene

KAR2 KAR2-2R: AGATGGGGTGATTCTGTTAC

TPO1-2F: CCTAACGACCCACTACATCC RT-qPCR for UPR downstream gene

TPO1 TPO1-2R: GCAGAGGCAAAAATGGAAGA

SIL1-2F ATCTGCGTTGATAATCGTTG RT-qPCR for UPR downstream gene

SIL1 SIL1-2R ATTCTAATGTCCAAACCACC

PDI1-F: TTACGAGGGACCTAGAACTG RT-qPCR for UPR downstream genes

PDI1 PDI1-R: ATTGGACGATAACTGGAGTG

ACT-F AGCTCCAATGAACCCTAAAT House-keeping gene ACT1 for

RT-qPCR ACT-R CGGAAGAGTACAAGGACAAA

Yeast medium and culturing conditions

Yeast cells were cultivated at 30°C in standard YPD (Yeast extract 10 g/L, Peptone

20 g/L, 2% glucose) or in synthetic medium (Yeast Nitrogen Base without amino acid

and ammonium sulphate 1.7g/L, ammonium sulphate 5g/L, 2% glucose) with

different supplement mixture according to the auxotrophs (minus histidine or uracil).

For solid medium 2% agar was added. Since Ga3+

and In3+

ions induce phosphate

metal precipitates in the normal synthetic medium (Schulze 2018), low phosphate

synthetic medium was prepared with Yeast Nitrogen Base without phosphate, and 64

mg/L KH2PO4 was added. In this way, fewer precipitates were formed to ensure a

higher concentration of free Ga3+

or In3+

ions in the medium. The stock solutions

(containing 100 mM ions) of Al2(SO4)3, InCl3 and GaCl3 were added into the medium

after autoclave to prepare synthetic medium plates with metal ions. The glycerol

11

stocks of yeast strains maintained at −80 °C were revived on YPD agar plates at 30 °C,

and the plates were stored at 4 °C for no more than 3 weeks.

Yeast transformation (short protocol)

Yeast cells were grown on YPD plates overnight at 30°C. The next day, a small

amount of yeast cells were taken with a loop and resuspended into 100 µL of

ONE-STEP buffer (40% PEG-4000, 0.2 M LiAc, 10 mM Tris, 1 mM EDTA). Then

2–5 µL plasmid DNA (500 ng–1 µg) and 1 µL recently denatured salmon sperm

ssDNA (10 mg/mL) were added. After mixing, the tubes were incubated at 42°C for

30 min. Then cells were centrifuged for 1min at 8000 rpm. The supernatant was

discarded, and the pellets were resuspended in 100µL PBS buffer (137 mM NaCl, 2.7

mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH adjusted to 7.4), and plated on

selective plates. The plates were incubated at 30 °C for 3 days.

Growth assay on solid medium

Yeast cells were pre-cultured overnight in liquid synthetic medium with different

supplement mixture at 30 °C and 200 rpm. The optical density (OD600) of the cell

culture was measured by 10 times dilution. The cell density was adjusted to OD = 1,

and 10-fold dilution series (OD600 = 1, 0.1, 0.01, 0.001) were prepared in 96-well

plates using a multichannel pipette. A small drop of liquid (~ 5 µL) from each dilution

was potted onto synthetic medium agar plates (with different supplement mixture

depending on the auxotrophic maker) with/without different metal ions. A sterilized

metal stamp was also used to pin the samples directly from the 96-well plates onto

agar plates. The plates were incubated at 30 °C for approximately 3–4 days to see the

spots, and the plates were checked every day.

RNA analysis

RNA extraction, cDNA synthesis and PCR assay

Around 1 × 107 yeast cells (approximately 2 mL of exponential phase/OD600 = 0.6–1

cell culture) were harvested in 1.5 mL tubes for RNA extraction. The cells were either

stored at −80 °C or directly disrupted for RNA extraction. Cells were mechanically

disrupted using the FastPrep-24™ instrument (MP Biomedicals) according to the

manufacturer's instruction specific for yeast cells. Total RNA was isolated using the

RNeasy Mini kit (Qiagen) according to the manufacturer's instructions for yeast. The

RNA concentration and quality were determined by a Nanodrop spectrophotometer

(Thermo Scientific). To remove contaminated DNA, 2 µg RNA in a total volume of

20 µL was used for DNase I (Thermo Scientific) digestion according to the manual.

Incubation was done in Thermal Cyclers from Bio-Rad. DNA digestion reactions

were terminated by EDTA and heat inactivation, and then the RNA samples were

used for cDNA synthesis without purification or stored at −80 °C.

10 µL of the DNA-free RNA from DNase I digestion mixture (containing 1µg

RNA) was used for cDNA synthesis. The Oligo dT primer and Maxima Reverse

Transcriptase (Thermo Scientific) were used according to the manufacturer’s

instruction (total cDNA synthesis reaction volume: 40 µL). The cDNA products were

stored without purification at −20 °C or −80 °C for a longer time. 2 µL of the cDNA

12

in a total volume of 20 µL reaction was used to carry out a 30-cycle PCR to measure

HAC1 mRNA splicing.

DNA digestion volume

DNAse Buffer (10X) 2 µL

DNAse I (1U/µL) 2 µL

RNA 2 µg

H2O up to 20 µL total

Incubated at 37 ºC for 30 min, then add EDTA (50 mM) 2 µL and incubated at 65 ºC for 10

min to terminate the digestion.

cDNA synthesis volume

After incubated at 65ºC for 5 min add:

5X RT Buffer 8 µL

Reverse transcriptase 2 µL

Incubated at 50 ºC for 30 min, then terminated by 65 ºC incubation for 5min.

Quantitative reverse transcription PCR (RT-qPCR)

RT-qPCR experiments were carried out using the TB Green® Premix Ex Taq ™ II

mixture (Takara) in a real-time PCR detection system (Bio-Rad). Sequences of

gene-specific primers were listed in Table 3. Reactions were performed with 1 µL of

1:4 diluted cDNA templates (generated from around 6.25 ng RNA) in a total volume

of 10 μl for each sample. The running condition was 1 min denaturation at 95 °C

followed by 40 cycles of 95 °C for 10 s and 57 °C annealing for 30 s. Nonspecific

amplicons were checked by dissociation curves. The amplification efficiency for each

primer pair was tested with a serial dilution of the original cDNA sample to 1/4, 1/8,

1/40, 1/80, 1/400 and 1/800.

The expression levels were normalized to that of the reference gene ACT1 which

encodes actin. Three technical replicates were analyzed to obtain the relative

expression quantity. The normalized ΔCT value relative to the non treated sample was

used to represent the expression change of the target gene (Livak and Schmittgen

2001). Results were presented as mean ± standard error (SEM). R studio was used for

statistical analyses. Data was analyzed using one-way ANOVA followed with

Dunnett’s test. Significant changes in expression levels were marked with * (p < 0.05),

** (p < 0.01) and *** (p < 0.001).

Oligo dT plus (10 µM) 8 µL

dNTPs (10 mM) 2 µL

H2O 10 µL

DNA-free RNA 10 µL

13

RT-qPCR volume

H2O 3.4 µL

SYBR Green Mix (2X) 5 µL

F primer (10 µM) 0.3 µL

R primer (10 µM) 0.3 µL

cDNA (1/4 dilution) 1 µL

Fluorescence microscopy

Yeast cells were pre-cultured overnight in synthetic liquid medium with different

supplement mixture in 10 mL tubes with lids. The next day, the pre-culture was

diluted 1:10 in the same medium and OD600 was measured. Then the pre-culture was

diluted into 50 mL medium with the initial OD600= 0.2. The cells were incubated for

4–5 h at 30 °C and 200 rpm to exponential phase (OD600= 0.6 or higher). Afterwards,

the cell culture was separated into 5 mL each in 50 mL falcon tubes, and metal ions

were added to the required concentrations. At the same time 1 mL non-treated cells

were collected as a negative control. The cells treated with metal ions were

continually incubated at 30 °C and 200 rpm. At different time points, 1 mL cell

culture of each sample was taken for fixation.

For cell fixation, 100 µL of 37% formaldehyde was added to 1 mL cells, mixed

and incubated for 30 min at room temperature. Afterwards, they were centrifuged for

1 min at 13000 rpm, and the supernatant was removed. Cells were resuspended and

washed twice with 1 mL PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,

2 mM KH2PO4, adjusted to pH 7.4 with NaOH or HCl). After the final wash, the cells

were resuspended in 15 µL PBS and stored at 4°C.

Cells were viewed at room temperature on an Axiovert 200M microscope (Carl

Zeiss MicroImaging) equipped with Plan-Apochromat 1.40-numerical-aperture

objectives (100×) and fluorescence light filter sets. Images were captured with a

digital camera (AxioCamMR3) and the AxioVision software, and processed with

ImageJ software for counting cells.

Results

Several yeast mutants were sensitive to Al3+

, Ga3+

and In3+

Some mutants were identified to be sensitive to Al3+

, Ga3+

and In3+

in yeast S.

cerevisiae through previous high throughput screening in our lab (Schulze 2018). In

this study, we first performed growth assays of 32 mutants from the screening on

synthetic medium with these metals to confirm the results. slt2Δ mutant was used as

the sensitive control strain (Hahn and Thiele 2002). As shown in Figure 4A, 4 mutants

(cnb1Δ, cch1Δ, pmr1Δ, slc1Δ) were hypersensitive to all the tested metals, among

which CNB1, CCH1and PRM1 are genes involved in metal homeostasis, while SLC1

is related to lipid metabolism. The deletion mutant of the phosphate hydrolysis related

gene PHO5 showed weak sensitivity to Al3+

and Ga3+

. Both sur1Δ and csg2Δ mutants

were only hypersensitive to In3+

, whereas SCT1 CST26 and ECM6 deletion mutants

14

were specifically sensitive to Ga3+

. UBP3-deleted mutant was sensitive to both Ga3+

and In3+

. Interestingly, sur1Δ and sct1Δ mutants showed totally different sensitivity to

Ga3+

and In3+

. The hac1Δ and ire1Δ mutants were very sensitive to Ga3+

, and also to

higher concentrations of In3+

(Figure 4B). HAC1 and IRE1 are key genes that mediate

UPR. All the sensitive mutants from the growth assay were summarized in Table 4.

Since we had the plasmid expressing IRE1 (pRS313-IRE1) with its native

promoter (Kimata, Oikawa et al. 2004)，we transferred the plamid to the ire1Δ mutant

to do the IRE1 complementation test. The ire1Δ mutant with pRS313-IRE1 showed

similar growth as the wildtype (WT), which confirmed that the IRE1 gene could

reverse the sensitive phenotype of the ire1Δ mutant (Figure. 4C). When introducing

the plasmid pA1 into the WT strain to overexpress ARR1 ARR2 and ARR3 genes

which are involved in arsenicals resistance (Bobrowicz, Wysocki et al. 1997), cells

showed no difference in growth at higher metal concentrations (Figure 4D),

suggesting the genes did not increase the tolerance of Ga3+

and In3+

in yeast.

A

B

15

C

D

Figure 4. Growth assay to confirm the sensitivity of mutants to Al3+

, In3+

and Ga3+

.

The wild type strain BY4741 (WT) and BY4741 slt2Δ were used as controls. Cells were pre-cultured in

synthetic medium, and 10-fold dilution series (OD = 1, 0.1, 0.01, 0.001) were spotted on synthetic

medium plates containing the indicated concentrations of Al3+

, In3+

or Ga3+

. The plates were incubated

at 30 °C 3 days until we saw the colonies. A. Growth assay of all the candidate mutants from high

throughput screening. B. Growth assay of hac1Δ and ireΔ1 mutants. Higher concentrations of Al3+

(5.5

mM) and In3+

(2.6 and 4 mM) were used to test the sensitivity. C. Growth assays to test the

complementation of IRE1. Wild type strain BY4741 with the empty plasmid pRS413, and ire1Δ mutant

with pRS313-IRE1 were cultivated in SC-HIS medium with metal ions as indicated. D. Growth assay

of BY4741 with empty plasmid pFL44L or plasmid pA1 for overexpression of ARR1, ARR2 and ARR3.

Cells were cultivated in SC-URA medium with metal ions as indicated. Higher concentrations of Al3+

(2.5 mM) and In3+

(2.2 mM) were used to test the tolerance.

Table 4. Summary of the sensitive mutants from growth assay

Metal Sensitive mutants

Al3+

Δcnb1, Δcch1, Δpmr1,Δslc1, Δpho5

In3+

Δcnb1, Δcch1, Δpmr1, Δ slc1, Δubp3, Δhac1,Δire1, Δsur1,Δcsg2,

Ga3+

Δcnb1, Δcch1, Δpmr1, Δ slc1, Δpho5, Δubp3, Δhac1,Δire1, ,Δsct1, Δcst26, Δecm6

UPR was unlikely induced by Ga3+

stress

Most secreted and membrane proteins go to the endoplasmic reticulum (ER) before

getting mature. Under stress conditions, unfolded proteins accumulate in the ER, and

as a result, the unfolded protein response (UPR) will be induced. Ire1 and Hac1 are

the main proteins that mediate UPR in yeast. When UPR is activated, the

transmembrane sensor protein Ire1 undergoes trans-autophosphorylation and

oligomerization. The HAC1 mRNA is then spliced by the RNase domain of the

activated Ire1 and translated into active transcription factor Hac1, which regulates

UPR target genes including ER chaperones, lipid biosynthesis enzymes and ERAD

(ER-associated degradation) machinery, to help protein folding and alleviate the ER

stress. (Shinjo, Mizotani et al. 2013; Mori 2015)

16

Our growth assay above showed the HAC1 and IRE1 deletion mutants were

hypersensitive to Ga3+

and moderately sensitive to In3+

, therefore we continued to test

whether Ga3+

induced UPR by checking the Ire1 clustering, HAC1 mRNA splicing

and activation of UPR down-stream genes.

Ire1 was not clustered in yeast cells exposed to Ga3+

Firstly we examined whether Ire1 was activated under Ga3+

stress. Previous studies

reported that Ire1 is activated through the formation of clusters (Kimata,

Ishiwata-Kimata et al. 2007). When the UPR is induced, the Ire1-GFP show a dot-like

distribution under fluorescence microscopy, whereas the unstressed cells show a

double ring-like distribution pattern which indicates that Ire1 dispersed throughout the

ER membrane (Kimata, Ishiwata-Kimata et al. 2007; Promlek, Ishiwata-Kimata et al.

2011). In this study, we used the same yeast strain (W303) with Ire1-GFP expression

in a plasmid (Le, Ishiwata-Kimata et al. 2016) to check the cellular localization of

Ire1 in cells exposed to Ga3+

for 1 h and 2 h. DTT which is a strong reducing agent

and blocks disulfide-bond formation to induce strong UPR activation was used as a

positive control treatment.

As shown in Figure 5, both the untreated cells and Ga3+

stressed cells showed a

double ring-like pattern of Ire1-GFP fluorescence, showing that Ire1 was on the

membrane, while the DTT treated cells showed a very clear dot-like pattern, showing

the Ire1 were clustered. Hence, we could not see clear Ire1 oligomerization and

activation under Ga3+

stress.

Figure 5. Cluster formation of Ire1.

W303 cells expressing Ire1-GFP were grown in synthetic medium to the exponential phase before

being treated either without or with 5 mM GaCl3 (Ga3+

) for 1 h and 2 h, or with 10 mM DTT for 1 h.

The cells were observed under fluorescence microscope. Three replicates with around 300 cells each

were checked for each treatment.

HAC1 mRNA was not spliced under Ga3+

stress

Then we checked whether Hac1 was activated under Ga3+

stress by measuring HAC1

mRNA splicing. Under ER stress, the RNase domain of activated Ire1 splices the

17

HAC1 mRNA precursor into mature mRNA, which is translated into functional

transcription factor protein Hac1 to induce various UPR target genes. Previous studies

showed that low concentrations of cadmium (Cd2+

) could induce HAC1 mRNA

splicing in yeast strain W303-1A, however, other toxic metals such as arsenite (As3+

)

and mercury (Hg2+

) did not exert such an effect (Gardarin, Chedin et al. 2010; Le,

Ishiwata-Kimata et al. 2016). In this study, BY4741 yeast cells were treated with 5

mM Ga3+

in normal synthetic medium or low phosphate medium (64 mg/L KH2PO4)

in which a higher concentration of free Ga3+

was expected. The reverse transcription

PCR was used to detect if the precursor form (p, 828 bp) of HAC1 mRNA changed to

the matured form (m, 576 bp). DTT was used as a positive control.

As shown in Figure 6, no spliced form of HAC1 mRNA was detected in cells

grown in both normal synthetic medium and the low phosphate synthetic medium,

when exposed to Ga3+

for 1 h, 2 h, 3 h, 4 h, or overnight. In contrast, HAC1 mRNA

was fully spliced in the positive control cells treated with 10 mM DTT for 1 h. Results

suggested the UPR maybe not induced by Ga3+

stress in the yeast strain BY4741.

A

B

Figure 6. HAC1 mRNA splicing measurement.

BY4741 cells were grown in synthetic medium (A) or low phosphate medium (B) to the exponential

phase before being treated either with 5 mM GaCl3(Ga3+

) for the indicated time (OV: overnight, about

12 h), or with 10 mM DTT for 1 h. After total RNA extraction, HAC1 mRNA splicing was detected by

reverse transcription PCR. Unstressed cells (Ctrl) and genome DNA (gDNA) were also used as controls.

The PCR products were separated using agarose electrophoresis and visualized with GelRed on a 1%

agarose gel. The position of the band corresponding to precursor (p) or mature (m) HAC1 mRNA is

indicated.

UPR-target genes were not up-regulated by a short time of Ga3+

stress

There are sets of genes up-regulated or down-regulated under ER stress, most of

which are related to ER protein folding, anti-oxidative stress or encode cell surface

proteins. Up-regulated genes including ER-located chaperones, protein folding

catalysts, ERAD factors and COP II components, while down-regulated genes mostly

encode cell surface or extracellular proteins (Travers, Patil et al. 2000). Here

18

quantitative reverse transcription PCR (RT-qPCR) was performed to analyze the

expression levels of four genes: KAR2 coding for ER chaperone; PDI1 responsible for

protein disulfide bond formation, SIL1 required for protein translocation into the ER,

and TPO1 coding plasma transmembrane protein. In the previous study, KAR2 PDI1

and SIL1 were drastically up-regulated under ER stress, while TPO1 was shown to be

significantly down-regulated (Kimata, Ishiwata-Kimata et al. 2006). The

amplification efficiency of all the primer pairs was evaluated using serially diluted

cDNA as templates, and a standard curve was drawn (Figure 7A). Correlation

between the cDNA concentration and Cq value suggested the amplification efficiency

of primers was between 99% and 109% which corresponds to a slope between −3.35

and −3.12, indicating they are good enough to perform the qPCR.

The BY4741 WT cells were treated with 5 mM Ga3+

in normal synthetic medium

or low phosphate medium for the indicated time. The fold changes of mRNA levels

were shown in Figure 7B and Figure 7C. In the normal synthetic medium (Figure 7B),

the mRNA levels of KAR2 PDI1 and SIL1 in cells treated with Ga3+

for 1 h and 2 h

were approximately similar to the non-treated controls. Whereas, the mRNA levels

increased dramatically (4–18 times) in response to the DTT treatment, which was

consistent with the previous study showing DTT is a strong UPR inducer (Le,

Ishiwata-Kimata et al. 2016). In low phosphate medium (Figure 7C), the mRNA

levels of KAR2 PDI1 and SIL1 in cells treated with Ga3+

for 1 and 2 h or with DTT

showed similar patterns as those in normal medium, indicating the UPR was only

induced by DTT. Increased mRNA levels of KAR2, PDI1 and SIL1 were found in

cells exposed to a longer time period of Ga3+

stress in low phosphate medium. For

instance, 4 h treatment of Ga3+

resulted in a slightly increased expression of PDI1,

while KAR2 PDI1 and SIL1 all showed a higher level of expression in response to

overnight of Ga3+

stress, which indicated a weak UPR activation (Figure 7C).

Interestingly, the TPO1 mRNA levels of all the treatments significantly decreased

compared with the untreated cells, which implied that TPO1 may be not suitable to

detect UPR.

19

A

B

23.5

23.6

25.7

26.4

29.6

30.8

y = -3.2868x + 29.369 R² = 0.9708

0

5

10

15

20

25

30

35

-1 0 1 2 3

Cq

Va

lue

Log concentration

KAR2

18.2

18.9

21.1

22.7

24.5

25.3

y = -3.1642x + 24.459 R² = 0.9929

0

5

10

15

20

25

30

-1 0 1 2 3

Cq

Va

lue

Log concentration

PDI1

23.6

24.1

26.3

27.2

29.6

30.6

y = -3.1242x + 29.556 R² = 0.9966

0

5

10

15

20

25

30

35

-1 0 1 2 3

Cq

Va

lue

Log concentration

SIL1

22.9

24.0

26.1

27.4

29.6

30.4

y = -3.2801x + 29.522 R² = 0.9987

0

5

10

15

20

25

30

35

-1 0 1 2 3

Cq

Va

lue

Log concentration

TPO1

16.0

17.1

19.3

20.3

22.5

24.0

y = -3.3581x + 22.716 R² = 0.9968

0

5

10

15

20

25

30

-1 0 1 2 3

Cq

Va

lue

Log concentration

ACT1

0

5

10

15

20

25

Control 1h 2h DTT

Fo

ld C

ha

ng

e R

ela

tive

mR

NA

Le

ve

ls

KAR2

***

0.0

1.0

2.0

3.0

4.0

5.0

Control 1h 2h DTT Fo

ld C

ha

ng

e R

ela

tive

mR

NA

Le

ve

ls

PDI1

***

0

2

4

6

8

10

12

Control 1h 2h DTT

Fo

ld C

ha

ng

e R

ela

tive

mR

NA

Le

ve

ls

SIL1

***

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Control 1h 2h DTT

Fo

ld C

ha

ng

e R

ela

tive

mR

NA

Le

ve

ls

TPO1

***

**

***

20

C

Figure 7. RT-qPCR monitoring mRNA levels of selected UPR downstream genes.

Yeast cells were grown in synthetic medium (B) or low phosphate medium (C) to the exponential

phase before being treated either with 5 mM GaCl3 (Ga3+

) for the indicated time (OV: overnight, about

12 h), or with DTT 10 mM for 1 h. After total RNA extraction, the mRNA expression was measured by

RT-qPCR analysis. ACT1 coding for actin was used as a house-keeping gene to normalize the

expression levels. The fold changes of mRNA levels of treatments to the mean of non treated cells

were shown. (Error bar: standard deviation) A. Evaluating of the amplification efficiency of each

gene-specific primer pair. The serially diluted cDNA was used as templates for qPCR, and slops of the

standard curve (the quantification cycles (Cq) vs the log of input DNA) was used for estimating the

amplification efficiency as indicated. B. RT-qPCR in normal synthetic medium. C. RT-qPCR in low

phosphate medium.

Measurement of other physiological changes under Al3+

, Ga3+

and In3+

stress

DNA damage

Our growth assay showed that mutants with deletion of genes involved in DNA repair

(RAD14, PSY3, and RDH54) were weakly sensitive to Ga3+

. To test whether Ga3+

or

In3+

induces DNA damage, we monitored the subcellular localization of the DNA

damage related proteins Rad52 and Rfa1 (Litwin, Bocer et al. 2013). Rad52 is a DNA

recombinase that repairs double-strand breaks (Symington 2002), and Rfa1 is a

subunit of the ssDNA-binding RPA complex involved in DNA replication (Grandin

and Charbonneau 2013). When there are double-strand breaks on the DNA, the YFP

tagged Rad52 and Rfa1 form distinct fluorescence foci which represent the DNA

repair center (Figure 8A). The previous study showed when treated with 0.5 mM As3+

for 1 h, around 10% of the cells showed distinct Rfa1 and Rad52 fluorescence foci,

0

5

10

15

20

25

Fo

ld C

ha

ng

e R

ela

tive

mR

NA

Le

ve

ls

KAR2

*

***

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Fo

ld C

ha

ng

e R

ela

tive

mR

NA

Le

ve

ls

PDI1

*** ***

*

0

1

2

3

4

5

6

7

Fo

ld C

ha

ng

e R

ela

tive

mR

NA

Le

ve

ls

SIL1

*

***

0.0

0.3

0.6

0.9

1.2

1.5

1.8

Fo

ld C

ha

ng

e R

ela

tive

mR

NA

Le

ve

ls

TPO1

***

** **

*

***

21

which was 2–3 folds increase compared with the non-treated control. (Litwin, Bocer

et al. 2013).

In our study, the same yeast cells expressing YFP tagged Rad52 or Rfa1 as the

previous study (Litwin, Bocer et al. 2013) were exposed to 2.6 mM In3+

or 5 mM Ga3+

for 1 h or 2 h. The fraction of cells with distinct damage foci was then quantified. For

non-treated control cells, we found similar percentages of cells with damage foci as

reported in the literature. However, the metal ion treated cells did not show a

significant increase in damage foci of both Rad52-YFP and Rfa1-YFP (Figure 8B).

A

B

Figure 8. Measurement of DNA damage under In3+

or Ga3+

stress.

A. Rad52-YFP and Rfa1-YFP form distinct fluorescence foci in nuclei as observed under microscope B.

Quantification of Rad52-YFP and Rfa1-YFP nuclear foci in cells treated with In3+

or Ga3+

. Standard

deviations are derived from three independent experiments (Student’s t-test.no significant difference, p >

0.05)

Al3+

and In3+

affected calcium homeostasis

The growth assay of our study showed the pmr1Δ, cch1Δ and cnb1Δ mutants were

hypersensitive to Al3+

, Ga3+

and In3+

. These three genes are involved in calcium

homeostasis: Pmr1 is a high affinity calcium ATPase which pumps cytosolic Ca2+

into

the ER and Golgi; Cch1 is a component of voltage-gated high-affinity calcium

channel involved in calcium influx (Stathopoulos-Gerontides, Guo et al. 1999); and

CNB1 encodes calcineurin B which is a Ca++

/calmodulin-regulated type 2B protein

phosphatase that regulates transcription factor Crz1 under stress (Cyert and Thorner

1992). Therefore we suspected Al3+

, In3+

, and Ga3+

influenced Ca2+

homeostasis,

which could be tested by monitoring the nuclear localization of GFP fused

0

0.5

1

1.5

2

2.5

%ce

lls w

ith

DN

A R

ep

air

Fo

ci

Rad52-YFP

0

1

2

3

4

5

6

%ce

lls w

ith

DN

A R

ep

air

Fo

ci

Rfa1-YFP

22

transcription factor Crz1. GFP-Crz1 was shown to gather in the nucleus when yeast

cells were incubated with 200 mM Ca2+

for 10 min in Crz1 deleted strain YPH499

(Stathopoulos-Gerontides, Guo et al. 1999).

In this study, BY4741 WT yeast cells expressing GFP-Crz1 in a plasmid were

exposed to 2.4 mM Al3+

2.6 mM In3+

or 5mM Ga3+

for 15 min and 30 min, and then

the fraction of cells with nuclear foci fluorescence (Figure 9A) was quantified. Under

Al3+

and In3+

stress for 15 min, a significantly higher percentage of cells showed

GFP-Crz1 nuclear foci, although the percentage of Crz1 activated cells then dropped

after 30 min (Figure 9B). The results indicated Al3+

and In3+

might influence Ca2+

homeostasis in yeast.

Phosphate homeostasis under metal stress

The growth assay in this study showed that the deletion mutant of phosphate

homeostasis related gene PHO5 displayed little sensitivity to Al3+

and Ga3+

, thus we

speculated the metal stress might also affect phosphate homeostasis. Therefore, we

assessed the nuclear localization of the GFP fused transcription factor Pho4, which

enters the nucleus to activate the transcription of downstream phosphate (PHO) genes

under phosphate limitation (O'Neill, Kaffman et al. 1996). A previous study showed

more than 90% of the cells had Pho4-GFP nuclear foci after phosphate starvation for

15 min, and Pho4 could go back into cytosol after re-addition of 10 mM phosphate

(Desfougeres, Gerasimaite et al. 2016).

Here the BY4741 WT yeast cells expressing Pho4-GFP were exposed to 2.4 mM

Al3+

, 2.6 mM In3+

, 5 mM Ga3+

or low phosphate medium (64 mg/L KH2PO4) for 30

min or 1 h, then the fraction of cells with nuclear fluorescence foci were quantified

(Figure 9A). As shown in Figure 9C, there was no significant difference in Pho4-GFP

nuclear foci formation between metal ion treated and the control cells. However, there

was a clear increase of Pho4-GFP nuclear localization in low phosphate treated cells

after 1 h.

A

23

B C

Figure 9. Nuclear localization of transcription factor Crz1 and Pho4.

BY4741 WT cells were transformed with a plasmid encoding GFP fused Pho4 or Crz1 under the

control of its own promoter. The localization of GFP-Crz1 and Pho4-GFP was assessed in living cells

cultured in synthetic medium with 2.4 mM Al3+

, 2.6 mM In3+

or 5 mM Ga3+

after 15 min 30min or 1 h.

For phosphate limitation treatments, cells were precipitated and suspended in low phosphate medium

(64 mg/L KH2PO4). The percentage of cells showing enrichment in nuclear Pho4 or Crz1 was

determined for these conditions. Around 900 cells (300 cells per replicate*3) were counted for each

condition. Standard deviations are derived from three independent experiments. (Student’s t-test. **, p

< 0.01. *, p < 0.05). A. Nuclear localization of GFP-Crz1 and Pho4-GFP in WT BY4741 under

fluorescence microscopy. B. GFP-Crz1 nuclear foci in Al3+

Ga3+

or In3+

treated cells. C. The nuclear

foci of Pho4-GFP in cells treated with Al3+

In3+

Ga3+

or limited phosphate. Lpho: low phosphate

treatment.

Discussion

Gallium and indium are rare elements widely used in electronics and energy industry.

They are not essential elements for living organisms and pose risks both to the

environment and human health. In this study, we aimed to understand their toxicity

mechanism at the molecular level using yeast S. cerevisiae.

Firstly, we confirmed the sensitivity of mutants identified from previous high

throughput screening and found some mutants were hypersensitive to Al3+

, Ga3+

and

In3+

, or specifically sensitive to In3+

or Ga3+

. Although all the candidate mutants were

shown to be sensitive in high throughput screening, only approximately one-third of

these mutants were verified by the growth assay. This was probably due to the

false-positive results in the high throughput screening, or different growth conditions

between the growth assay and the previous high throughput study. For instance, the

cells in high throughput screening had smaller growth areas and were exposed to

more stress which resulted in higher sensitivity. Besides, the cell growth in high

throughput screening was monitored by machine, which was less accurate than the

manually growth assay we did here.

Contrary to what we expected, we did not find Ga3+

stress induced UPR when

cells were stressed with GaCl3 for hours (until 4 h), despite the clear sensitivity

displayed by the UPR pathway mutants (hac1Δ and ire1Δ) in the growth assay, and

0

1

2

3

4

5

6

7

8

9

10

%ce

lls w

ith

Crz

1-G

FP

Nucle

ar

Fo

ci

Crz1-GFP

**

** *

0

2

4

6

8

10

12

14

16

18

%ce

lls w

ith

En

rich

ed

Ph

o4

Pho4-GFP

24

even though the IRE1 expressing plasmid could complement the phenotype of the

ire1Δ mutant. We did not detect the activation of Ire1, and did not observe HAC1

mRNA splicing, furthermore, the UPR target genes showed no significant

up-regulation after treated with Ga3+

for 4 h. Previous HAC1 mRNA splicing was

measured mostly in yeast strain W303 which was more sensitive to stress (Kvitek,

Will et al. 2008), whereas we used BY4741 since the screening was done in this strain.

In the following experiments, we could also test if Ga3+

will trigger HAC1 mRNA

splicing in W303 to further confirm the results obtained.

The mRNA level of UPR target genes started to rise after 4 h of Ga3+

stress in low

phosphate medium, however previous studies showed that the UPR activation could

be induced by metal ions in a shorter period of time (the usual exposure time is 1 h)

(Le, Ishiwata-Kimata et al. 2016). Therefore we believe the up-regulation of the genes

was probably not induced by metals, but due to the physiological changes related to

cell growth. When we added InCl3 or GaCl3 into the synthetic medium, the

precipitates emerged immediately, and the pH of the medium dropped dramatically to

2–3. Our previous work revealed that most of the precipitates were phosphate metal

compounds. Thus, the addition of these two metals ions changed the medium

composition such as pH and phosphate concentration, which could influence the

growth of the mutants, especially in the long term cultivation (more than 4 h).

Alternatively, it’s possible to grow the cells in YPD medium with In3+

and Ga3+

, since

it contains more organic phosphate and may cause fewer precipitates. However, the

composition of YPD is complex, which also makes it difficult to interpret the results.

Finally, we checked whether Al3+

, Ga3+

or In3+

stress affected other cellular

functions such as DNA damage, calcium and phosphate homeostasis. We found Al3+

or In3+

stress could influence calcium homeostasis. The calcium homeostasis

experiment needs to be verified further since we did the experiment in a different way

as the previously reported study. The study before used a Crz1 deleted strain YPH499

expressing GFP-Crz1 in a plasmid to test the Ca2+

induced nuclear translocation

(Stathopoulos-Gerontides, Guo et al. 1999). Here we used the WT BY4741 strain

transformed with the plasmid, and we were not sure if the presence of native Crz1 will

affect GFP-Crz1. Besides, in the previous study, the cells were treated with Ca2+

at

21 °C, whereas all of our treatments were conducted at 30 °C. It is also necessary to

include Ca2+

treatment as a positive control, notably the ammonium sulfate should be

replaced by ammonium chloride to avoid CaSO4 precipitates. Similarly, in the

phosphate homeostasis measurement experiment, it is better to use phosphate

depletion treatment as a positive control. Here we used low phosphate medium (64

mg/L KH2PO4) equivalent to 0.47 mM phosphate as a positive control, whereas the

literature showed 10 mM phosphate would totally recover the phenotype and Pho4

went back to cytosol. As cells grow, the available phosphate in the medium becomes

less, and probably that is why our results showed the Pho4-GFP nuclear foci rise after

1 h, but in the previous study, only 15 min phosphate starvation was needed to see 90%

cells with nuclear foci.

We also considered measuring the intracellular metal concentrations of metal ions

stressed yeast cells, yet the method was to expose the cells in the liquid synthetic

25

medium where In3+

or Ga3+

cause precipitates that continually accompany with yeast

cells and cause false positive results. An alternative way for future study is to culture

the strains in the solid medium, so the precipitates will be left in the plates when

harvesting the cells, or to centrifuge the liquid medium after adding metal ions, and

remove the precipitates before cell cultivation.

Conclusion

This study verified that some of the deletion mutants of yeast Saccharomyces

cerevisiae BY4741 were sensitive to Al3+

, Ga3+

and In3+

, among which the deleted

genes were mainly related to metal homeostasis (cnb1Δ, cch1Δ, pmr1Δ), lipid

metabolism (slc1Δ) and UPR (hac1Δ, ire1Δ). Further investigation found Ga3+

stress

did not induce significant UPR, whereas Al3+

and In3+

might influence calcium

homeostasis. Although the background mechanism was still not clear, the results still

provide valuable suggestions for future study. The study of Al3+

stress in yeast is

easier to continue and may provide some clues for the In3+

and Ga3+

toxicity. Until we

develop a method to exclude the influence of precipitates by In3+

or Ga3+

, it is still

challenging to illustrate the molecular mechanism.

Acknowledgment

I feel lucky to work in Markus Tamas lab and I am really thankful to him for

providing the opportunity and support to do this interesting thesis project.

I am thankful to Joana who spent a lot of time sharing her experience in the lab,

which was really useful for me to get started at the beginning. I would also like to

thank Antonia who helped me a lot with RNA extraction and RT-qPCR, and also other

people from the lab that helped me.

I am thankful to my husband and daughter who make my life meaningful.

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