Stress-seventy subfamily A 4, A member of HSP70, confers yeast cadmium tolerance in the loss of mitochondria pyruvate carrier 1

ABSTRACT

Mitochondrial pyruvate carrier (MPC), which transports pyruvate into mitochondria, is a key regulatory element in the material metabolism and energy metabolism. Since MPC was firstly identified in yeast in 2012, many groups have investigated the function of MPC. As MPC is a classic material transporter, the focus of previous studies has been placed on its role in pyruvate transport. In this study, we discovered a novel Cd resistant gene, stress-seventy subfamily A 4 (SSA4), which can recover the Cd sensitive phenotype in the yeast MPC1 mutant strain. It is suggested that, except for adjusting metabolism, MPC can regulate stress tolerance by regulating downstream genes in yeast. Previously, we discovered a Cd related gene, AGP30, which is associated with MPC1 in Arabidopsis. These results indicate that MPC can regulate Cd tolerance through downstream genes in both Arabidopsis and yeast. This study will pave the way for further exploring the bypass pathways of MPC at the molecular level, and the interaction between MPC and the downstream genes in biology.

Mitochondrial pyruvate carriers (MPCs), which are the critical regulation elements for both substance and energy metabolisms, were firstly identified in Drosophila and yeast.^1,2 Previously, we found that MPC1 is required for Cd tolerance in Arabidopsis and yeast. In Arabidopsis, MPC prevents Cd toxicity by sustaining TCA cycle and ATP, and alleviates the pressure of GSH synthesis.³ Recently, AGP30, a Cd tolerance related gene, was also proven to be involved in Cd tolerance associated with AtMPC1 in Arabidopsis. When treated with Cd, the transcript abundance of AGP30 decreased by half; without AtMPC1, the abundance dropped over 50 fold.⁴ These results indicate that the function of MPC in Cd tolerance regulation is complex, and there exist downstream candidate genes which associate Cd stress in Arabidopsis. In yeast, the mutant strain Δmpc1 also shows a sensitive phenotype compared with the wild-type strain JRY472 when treated with 50 μM Cd. Although MPC shares similar metabolic and Cd resistance mechanisms in Arabidopsis and yeast, it is unclear whether other molecular bypass pathways exist in regulating Cd toxicity in yeast under the control of MPC. To verify this, RNA-seq analysis was conducted on Cd-treated yeast cells comparing Δmpc1 against the wild-type strain (JRY472) (Figure 1(a)). Differentially expressed genes (DEGs) were identified with good repeatability from three biologically independent repeats (adjusted P value < .05), and log2 ratio >0.7 or <-0.7 were selected for the following analysis (Supplementary Table 1). Out of the DEGs, 1023 genes were up-regulated and 1003 genes were down-regulated.

Figure 1.

RNA-seq analysis. (a) Yeast wild type strain and mpc1Δ cells were grown at 30°C in YPAD liquid media and exposed to 50 µM CdCl₂ at the concentration of 0.3 OD₆₀₀. Cell density was monitored with the absorbance at 600 nm at 15, 18, 21, 24 h after treatment. Error bars indicate ± SD from three independent experiments. After centrifuging 30s at 12000g, the yeast cells in tubes were instantly freezed in liguid nitrogen, and then stored in −80°C. (b) Numbers of DEGs in the comparison group (Δmpc1-1 VS Wt). (c) The most 30 enriched GO terms in the comparison group (Δmpc1-1 VS Wt). * represent the corrected P value of the term < 0.05 in GO term enrichment analysis. (d) The most enriched KEGG pathways in the comparison group (Δmpc1-1 VS Wt). Rich factor indicates the ratio which the DEG number compare to the background number in the enriched KEGG pathway. qvalue is Pvalue after multiple hypothesis test correction.

To understand the molecular regulatory mechanism, Gene Ontology (GO) enrichment analysis of the DEGs was primarily carried out by the GOseq.⁵ As shown in Figure 1(c) and Supplementary Table 2, 20 pathways were enriched, including pathways in biological process, cellular component and molecular function. Out of the enriched pathways, most are related to metabolism or material synthesis, and there is no term describing heavy metal stress or oxidative stress. Considering there are many terms related to heavy metal or oxidative stress in the GO database, our result suggested that the Cd tolerance function of MPC could be not due to the classical heavy metal or oxidative stress tolerance. We also conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. As shown in Figure 1(d), most of the pathways were involved in material metabolisms. These results indicated that the Cd sensitive phenotype caused by the loss of MPC1 in yeast is due to the interruption of some metabolic pathways or material pathways.

To test whether downstream candidates were involved in the Cd stress tolerance mechanism regulated by MPC1, four most significantly down-regulated DEGs (SPG1, MEP2, SSA4, and YGR035C) were selected (Table 1). The SPG1 is required for high temperature survival during stationary phase, and detected in highly purified mitochondria in high-throughput studies.^6,7 As an ammonium transport protein, MEP2 regulates pseudohyphal differentiation and is controlled by phospho-silencing.^8,9 SSA4 is a member of Heat Shock Protein 70 (HSP70) gene family.^10–12 Although HSP70 members can be induced and associate with heavy metal stress, ^13–15 there is no evidence indicating that SSA4 confer Cd tolerance in any species. YGR035C whose transcription is activated by paralogous transcription factors Yrm1p and Yrr1p along with genes involved in multidrug resistance is potential Cdc28p substrate and uncharacterized.^16,17 After the four genes were cloned into the yeast expressing recombination vectors (pGPD::DEGs) and yeast transformation, a serial dilution assay was conducted to test Cd tolerance. As shown in Figure 2, Stress-seventy subfamily A 4 (SSA4) can partially recover the Cd sensitive phenotype of ⊿mpc1. Our results indicate that, except for metabolic regulation, MPC1 also regulated Cd tolerance through SSA4, which is a downstream gene in the MPC1 controlled Cd resistant pathway. Previously, we reported that MPC prevents Cd toxicity by sustaining the TCA cycle and glutathione synthesis. We also discovered a resistant gene, AGP30, which is related to Cd tolerance and associated with MPC1. It is suggested that MPC1 plays a role in Cd tolerance through regulating metabolism and downstream gene pathways in Arabidopsis. In this study, we found MPC1 was also required for Cd tolerance through regulating metabolism and downstream Cd tolerance genes in yeast. Although MPC is a classic key material transporter, it could not affect downstream genes by direct interaction, however, under Cd stress, MPC can confer the tolerance associating with some specific gene. The function of MPC in molecular regulatory pathways needs to be further explored.

Table 1.

The most significant down-regulated differentially expressed genes (DEGs) (Log2 (Fold Change) < −4) in Δmpc1 VS WT comparison group discovered by RNA-seq.

Gene_ID	Read Count (Δmpc1)	Read Count (WT)	Log2 (Fold Change)	P value	Corrected P Valve	Associated Gene Name
YGR236C	68.05330935	2347.520994	−5.1083	3.78E-06	5.37E-05	SPG1
YNL142W	719.9926712	16594.1596	−4.5265	1.69E-35	3.76E-32	MEP2
YER103W	1935.029646	41848.86568	−4.4348	6.84E-41	2.28E-37	SSA4
YGR035C	353.1023624	5883.860286	−4.0586	9.08E-35	1.51E-31	-//-

Figure 2.

Yeast dilution bioassay with wild-type strain, mpc1Δ transformed with pRS416 and pRS416 expressing ScMPC1 in SC medium. Triangles represent serial 10-fold dilutions (starting concentration of 0.3 OD₆₀₀). Representative test from three reproducible experiments was shown.

Funding Statement

This study was supported by National Key Research and Development Program of China [grant no. 2017YFD0101801], Modern Agricultural Industrial Technology System Funding of Shandong Province, China [grant no. SDAIT-05-04], Shandong Upgraded Project of “Bohai Granary” Science and Technology Demonstration Engineering in 2019, the Special Foundation for Postdoctoral Innovation Projects under Grant 201603036, Taishan Scholars Program of Shandong Province (No.tsqn201909167).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.