Global Deletome Profile of Saccharomyces cerevisiae Exposed to the Technology-Critical Element Yttrium

Abstract

The emergence of the technology-critical-element yttrium as a contaminant in the environment raises concern regarding its toxicological impact on living organisms. The molecular mechanisms underlying yttrium toxicity must be delineated. We considered the genomic phenotyping of a mutant collection of Saccharomyces cerevisiae to be of particular interest to decipher key cellular pathways involved either in yttrium toxicity or detoxification mechanisms. Among the 4733 mutants exposed to yttrium, 333 exhibited modified growth, of which 56 were sensitive and 277 were resistant. Several functions involved in yttrium toxicity mitigation emerged, primarily vacuolar acidification and retrograde transport. Conversely, functional categories overrepresented in the yttrium toxicity response included cytoskeleton organization and endocytosis, protein transport and vesicle trafficking, lipid metabolism, as well as signaling pathways. Comparison with similar studies carried out using other metals and stressors showed a response pattern similar to nickel stress. One third of the identified mutants highlighted peculiar cellular effects triggered by yttrium, specifically those affecting the pheromone-dependent signaling pathway or sphingolipid metabolic processes. Taken together, these data emphasize the role of the plasma membrane as a hotspot for yttrium toxicity. The up-to-now lack of data concerning yttrium toxicity at the cellular and molecular levels makes this pioneer study using the model S. cerevisiae an excellent first basis for the assessment of yttrium toxicity toward eukaryotes.

Keywords: Saccharomyces cerevisiae; genome-wide screening; technology critical element; yeast mutants; yttrium toxicity.

FIGURE 1

Representative primary screen data for yttrium-responsive mutants and phenotypic confirmation of the identified mutants. (A) Representative 384-well format growth test for the yttrium toxicity primary screen (four pin replication of each mutant). A control plate (YPD medium) and the same plate supplemented with 3.75 mM yttrium are shown. Putative yttrium sensitive (blue) and resistant mutants (orange) are highlighted. (B) Phenotypic confirmation of selected sensitive and resistant mutants. Wild-type and mutant strains were grown without or with yttrium. Yttrium sensitivity was determined by 10-fold serially diluted spot assays (left to right) with saturation phase grown cells. Mutant strains exhibiting a reduction in colony-forming ability at the first, second-third or fourth-fifth dilution were classified as “high” (HS), “medium” (MS), or “low” (LS) sensitive to yttrium, respectively. Conversely, mutant strains exhibiting an increase in colony-forming ability at the second, third-fourth, or fifth-sixth dilution were classified as “low” (LR), “medium” (MR), or “high” (HR) resistant to yttrium, respectively.

FIGURE 2

Distribution and ranking of yttrium-responsive mutants. The middle pie chart shows the overall number of resistant (orange) and sensitive (blue) mutants. Detailed proportions of the different categories low (LR), medium (MR), high (HR) resistant mutants (left) and low (LS), medium (MS), and high (HS) sensitive mutants (right) are shown. ORF names of HR and HS mutants are mentioned.

FIGURE 3

In silico comparison of phenotypes of the mutants identified in this study compared with those from other metal-based screening studies. Different colors represent the mean phenotype of the clustered mutants (orange: resistant, blue: sensitive). Functions overrepresented in the clusters (C1–C10) are mentioned on the right. The number of mutants within each cluster or function is specified in brackets. Functions considered were retrieved from FunSpec (biological process GO terms, P < 0.01). The hierarchical clustering was done with the following parameters: average linkage, uncentered correlation, k-mean = 10.

FIGURE 4

Hierarchical clustering of yttrium sensitivity or resistance-conferring mutations with the mutant sensitivity/resistance profiles of other stressors. The x-axis corresponds to gene deletions, and the y-axis represents the different physico-chemical stressors. Mutant strains exhibiting either a higher sensitivity, a higher resistance, or no phenotype change when compared to wild-type are shown in blue, orange, and gray, respectively. Non-metal stressors were selected from previous genomic phenotyping screenings conducted on deletion mutant collections. Methyl methane sulfonate (MMS), gamma-radiation (γ-ray), alkaline pH (pH), menadione (Men), hydrogen peroxide (H₂O₂), cumene hydroperoxide (CHP), linoleic acid 13-hydroperoxide (LoaOOH), and diamide (DM). Hierarchical clustering was done with the following parameters: average linkage and uncentered correlation.

FIGURE 5

Functional enrichment analysis network of functions that when deleted render cells either sensitive (blue nodes) or resistant (orange nodes) to yttrium. Green lines represent gene overlap between two functions, with the edge width being proportional to the number of shared genes. The enrichment map was built using GSEA and visualized by the enrichment map plugin in Cytoscape.

FIGURE 6

Global representation of yttrium sensitive/resistant mutants involved in the endocytic pathway in S. cerevisiae. (A) Schematic representation of the endocytic pathway highlighting the yttrium response of KO-mutants involved in the different associated steps. It includes endocytosis (1), targeting to (and formation of) the multivesicular body (MVB) compartment (2), protein retrieval from the MVB to the late Golgi by the Retromer and GARP complex (3), Golgi-to-vacuole trafficking (4), and MVB-to-vacuole fusion (5) pathways. Blue arrows denote the pathways in which the lack of a given protein render cells sensitive to yttrium, while orange arrows correspond to yttrium-resistant mutants. (B) Ten-fold dilution drop test assays of mutants for proteins involved in the different mentioned steps (1–5). For the color code of the level of sensitivity or resistance, please refer to the legend of Figure 1.