MCHM Acts as a Hydrotrope, Altering the Balance of Metals in Yeast

Abstract

While drugs and other industrial chemicals are routinely studied to assess risks, many widely used chemicals have not been thoroughly evaluated. One such chemical, 4-methylcyclohexane methanol (MCHM), is an industrial coal-cleaning chemical that contaminated the drinking water supply in Charleston, WV, USA in 2014. While a wide range of ailments was reported following the spill, little is known about the molecular effects of MCHM exposure. We used the yeast model to explore the impacts of MCHM on cellular function. Exposure to MCHM dramatically altered the yeast transcriptome and the balance of metals in yeast. Underlying genetic variation in the response to MCHM, transcriptomics and, mutant analysis uncovered the role of the metal transporters, Arn2 and Yke4, to MCHM response. Expression of Arn2, which is involved in iron uptake, was lower in MCHM-tolerant yeast and loss of Arn2 further increased MCHM tolerance. Genetic variation within Yke4, an ER zinc transporter, also mediated response to MCHM, and loss of Yke4 decreased MCHM tolerance. The addition of zinc to MCHM-sensitive yeast rescued growth inhibition. In vitro assays demonstrated that MCHM acted as a hydrotrope and prevented protein interactions, while zinc induced the aggregation of proteins. We hypothesized that MCHM altered the structures of extracellular domains of proteins, and the addition of zinc stabilized the structure to maintain metal homeostasis in yeast exposed to MCHM.

Keywords: Hydrotrope; Ionome; Iron; MCHM; Mitochondria; Petite; Yeast; Yke4; Zinc; Zrt1.

Fig. 1

Schematic of yeast cellular response to varying levels of zinc. Under low intracellular zinc levels, Zap1 induces expression of zinc uptake genes. Zrt1, the high-affinity, and Zrt2 the low-affinity transmembrane zinc transporters are localized in the cell membrane. Izh1 and Izh4 localize to the cytoplasmic side of the cell membrane and regulate zinc homeostasis. Excess zinc (blue hexagons) is stored in the vacuole and transported in by Zrc1 and Cot1. Excess zinc stored in the vacuole, and when needed, Zrt3 transports zinc to the cytoplasm. In addition to the vacuole, the ribosome (red circles) bind 20% of total cellular zinc [14]. Yke4 is the transmembrane transporter at the ER (pink and purple) that transports zinc in both directions. Sod1 is the copper-zinc super oxygen dismutase that is both cytoplasmic and localizes to the inner mitochondrial membrane

Fig. 2

Serial dilution of wild-type S96 and petite yeast in increasing concentration of MCHM in rich (YPD) and minimal (YM) media. Plates were incubated at 30 °C for 3 days and then photographed

Fig. 3

Changes in RNA expression between wild type, petite grown in YPD treated with MCHM. The number of up and downregulated genes is noted on the top of each panel. a Scatter plots of log fold 2 comparisons of RNA-seq from grande (S96) and petite (S96ρ) yeast grown in YPD. Significantly upregulated genes are labeled in red and significantly downregulated genes are labeled in blue. b Scatter plots of log fold 2 comparisons of RNA-seq grande yeast grown in YPD and with 550 ppm MCHM. c Scatter plots of log fold 2 comparisons of RNA-seq from petite yeast grown in YPD and with MCHM. d Scatter plots of log fold 2 comparisons of RNA-seq from S96 and S96 yeast grown in YPD and with MCHM

Fig. 4

Measurements of metals in yeast treated with MCHM. a Levels of Fe, Zn, Ca, Na, Mg, K and K in μg/mg from grande (S96) and petite (S96ρ) yeast grown in YPD. Levels of metals from grande (S96) and petite (S96ρ) yeast grown in YPD with 550 ppm MCHM added for the indicated time. The ions levels measured were b Fe, c Zn, d Ca, e Na, f Mg, g K, and h F. The standard error is noted on the mean of four biological replicates

Fig. 5

Genetic variation of MCHM response linked to the YKE4 locus. a Serial dilution of genetically diverse yeast on 550 ppm MCHM on YPD. S96 are from the S288c background and YJM789 is a clinical isolate. b Quantitative trait loci analysis of chromosomal regions linked to increased growth of yeast in MCHM from YJM789 and S96 segregants. c Diagram of Yke4 describes the transmembrane domains (TM noted with gray box) and inside or outside domains (noted with lower dark gray or upper light gray lines, respectively). Polymorphic residues are noted below in Yke4^YJM789 compared to Yke4S288c. The conserved ZIP domain is boxed in a gray dashed line. d Serial dilutions of S96 and YJM789 with yke4 mutants on MCHM. e Levels of total intracellular zinc from S96, YJM789, and yke4 mutants normalized to S96 grown in YPD and total protein. Yeast were incubated with 5 μM zinc sulfate, 550 ppm MCHM, and in combination for 30 min before metals were extracted

Fig. 6

Impact of loss of metal transporters on growth with MCHM and zinc-containing media. a Serial dilution of BY4741 and yeast from the knockout collection on YPD with 400 ppm MCHM. b Serial dilution of wild-type (BY4741) and zinc transporters knocked out yeast grown in 400 or 550 ppm MCHM on YPD with 10 μM zinc sulfate

Fig. 7

MCHM role in protein aggregation. a Chemical structure of the cis and trans conformation of MCHM. b Timed protein aggregation when exposed to heat. No treatment is compared to 10 mM sodium xylene sulfate, 1 mM ATP, and 550 ppm MCHM. The optical density of samples was measured at 450 nm after incubation at 60 °C. 550 ppm MCHM was added to samples then 10 μM zinc sulfate was added where indicated. c Spheroplast yeast were incubated for between 0 and 15 h with 0.1% SDS, 0.5% water, 10 mM sorbitol, 1 mM ATP, 10 mM NaXS, 10 μM zinc sulfate and 550–1000 ppm MCHM, and samples were read at 600 nm. Biological quadruplicates were averaged and standard error. Relevant p values were calculated using the student t test