Shine et al. 2015 — Phytochelatin synthase (PCS1) is required for Cd and Cu tolerance in a basidiomycete yeast and is broadly conserved across fungi

Shine, Shakya, and Idnurm (Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri–Kansas City; School of BioSciences, University of Melbourne) identify a putative phytochelatin synthase gene (PCS1) in the basidiomycete yeast Sporobolomyces sp. strain IAM 13481 (Pucciniomycotina), demonstrate by heterologous expression in Saccharomyces cerevisiae and Schizosaccharomyces pombe that it confers Cd tolerance, generate a pcs1Δ deletion in Sporobolomyces that is hypersensitive to cadmium and copper (and slightly to zinc), show by GFP fusion in S. pombe that Pcs1 co-localises with the mitochondrial dye MitoTracker, and use comparative genomics of 375 fungal genome sequences across 18 lineages and sub-lineages (Table 1 sum) to map a wide but punctate distribution of PCS homologs across the fungal kingdom (present in most early-diverging phyla, lost in much of the Dikarya including all Saccharomycotina and Agaricomycotina). The paper extends the function of PCS — previously characterised in only two ascomycete species (S. pombe and Tuber melanosporum) — into a second major fungal lineage, and argues that PCS is an ancestral eukaryotic metal-homeostasis factor that has been lost repeatedly in fungi, parallelling the distribution seen in plants and animals. Published in Fungal Biology and Biotechnology 2:3, 28 March 2015 (received 11 Nov 2014; accepted 11 Mar 2015). In the wiki this is mechanistic background for cadmium (PCS-mediated detoxification in fungi), copper (PCS role in essential-metal homeostasis), and zinc; it contains no food-matrix, environmental, or human-exposure measurements.

Why this matters

• It establishes that PCS is functional in the Basidiomycota, not just the Ascomycota. Before this paper, fungal PCS function had been characterised in only two species, both ascomycetes: Schizosaccharomyces pombe (Clemens et al. 1999; Ha et al. 1999) and the truffle Tuber melanosporum (Bolchi et al. 2011). The Sporobolomyces result places PCS function in a second major fungal lineage (Pucciniomycotina, Basidiomycota), supporting the framing that PCS is an ancestral eukaryotic gene rather than a plant-derived horizontal transfer into a handful of fungi.
• It shows that the fungal PCS protein is involved in both non-essential-metal detoxification (Cd) and essential-metal homeostasis (Cu, and to a lesser extent Zn). The Sporobolomyces pcs1Δ mutant is hypersensitive to cadmium and to copper (Figure 2D), and the S. pombe pcs1Δ mutant shows both Cu growth sensitivity and a pigmentation phenotype indicative of altered Cu homeostasis (Figure 3B). This complicates the legacy framing of PCS as a heavy-metal-detoxification gene only; for the wiki’s copper page, it is one of the experimental backbones for the view that PCS-type chelation has a maintenance role in essential-metal homeostasis, not only an emergency role in non-essential-metal toxicity.
• It demonstrates that PCS proteins localise to the mitochondria in S. pombe. Pcs1-GFP co-localises with MitoTracker red (Figure 4B), and computational subcellular-localisation predictors (PSORT II, MitoProt) identify N-terminal mitochondrial targeting sequences in PCS homologs from organisms as evolutionarily distant as Arabidopsis thaliana and Caenorhabditis elegans. Prior reports had localised the helminth Schistosoma mansoni PCS to the mitochondria (Ray et al. 2011; Rigouin et al. 2013), and the Arabidopsis AtPCS1 has been reported in the cytoplasm (Blum et al. 2010); this paper adds a fungal data point and contributes to a still-open question on PCS subcellular distribution that is relevant to any future wiki section on cellular Cd handling.
• It maps PCS distribution across the fungal kingdom from 375 genome sequences (Table 1 sum across nineteen lineage/sub-lineage rows) and finds the gene is broadly distributed in early-diverging fungal phyla (Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Glomeromycota, Mucoromycotina, Kickxellomycotina, Wallemiomycetes, Cryptomycota, Monoblepharidomycetes) but absent from large portions of the Dikarya — entirely absent from Saccharomycotina (35 genomes), Agaricomycotina (103 genomes), and Ustilaginomycotina (8 genomes), and present in the Pezizomycotina (180 genomes examined) only within the class Pezizomycetes (Table 1). The authors interpret this as multiple independent gene losses from a widely-distributed ancestral state rather than sparse horizontal transfer. For the wiki, this conservation pattern is the reason to treat PCS as a default eukaryotic metal-homeostasis factor when describing what fungal communities can and cannot do with metals in food/agricultural/soil systems.
• The Sporobolomyces model is itself notable for the metals literature: this yeast was isolated from the leaf surface of a willow tree (not from a contaminated site), establishing that PCS function is selected for under ordinary environmental Cd/Cu/Zn loads, not only under industrial pollution exposure. Pucciniomycotina yeasts have separately been isolated from high-Cd/Cu metal sites (cited as the basis for the “primary role in essential-metal homeostasis” hypothesis the discussion proposes).

Key numbers

All values reported in the source’s own units; no conversions applied. Concentrations are given as the metal salt molarity that the source used (CdSO₄, CuSO₄, ZnSO₄, etc.). For Cd, 1 µM CdSO₄ contains 1 µmol Cd/L ≈ 112 µg Cd/L (Cd atomic mass 112.41 g/mol).

Strains used (Table 2). Sporobolomyces sp. wild type IAM 13481; ura5 auxotroph AIS2; pcs1Δ::URA5 ura5 deletion AS1 (intermediate, not used for assays); pcs1Δ::ura5 ura5 AS2 (the assay deletion strain after URA5 recycling on 5-FOA); pcs1Δ ura5 ura5 + PCS1-URA5 complemented control AS3. S. cerevisiae BY4743 (MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0); yap1::KanMX in BY4743 background; S. cerevisiae strains AS4–AS7 expressing pYES2 empty or pAS1 (Sporobolomyces PCS1 under GAL1) in WT or yap1::KanMX backgrounds. S. pombe L972 wild type and MM72-4A ura4-D18 mutant; pcs1::KanMX ura4 deletion AS8 (parent MM72-4A). Per Table 2 (p. 9), the AS9–AS11 trio carries the MM72-4A (WT ura4-D18) background and serves as the wild-type-background control panel: AS9 = MM72-4A + empty pREP42, AS10 = MM72-4A + S. pombe PCS1 (pAS4), AS11 = MM72-4A + Sporobolomyces PCS1 (pAS5). The AS12–AS14 trio carries the AS8 pcs1Δ background and is the assay panel for complementation: AS12 = pcs1Δ + empty pREP42, AS13 = pcs1Δ + S. pombe PCS1 (pAS4), AS14 = pcs1Δ + Sporobolomyces PCS1 (pAS5). AS15 = pcs1Δ + PCS1-GFP (pAS6) is the GFP-fusion derivative of AS8 used for microscopy and the Cd-complementation rescue in Figure 4A.

Heterologous expression of Sporobolomyces PCS1 in S. cerevisiae (Figure 1). Ten-fold serial dilutions of WT or yap1Δ carrying empty pYES2 or pAS1 (PCS1 under GAL1 promoter), cultured overnight in YNB + glucose or YNB + galactose, plated on YPD + 0 / 10 / 100 µM CdSO₄, 2 d at 30°C. Sporobolomyces PCS1 increased Cd resistance of both WT and yap1Δ strains on both 10 µM and 100 µM Cd. The increase was visible even under non-inducing glucose conditions, indicating that residual GAL1 expression provides enough Pcs1 to combat Cd toxicity.

Sporobolomyces pcs1Δ (AS2) metal-stress panel (Figure 2D and text on p. 2, second column). Ten-fold serial dilutions of WT, pcs1Δ, and Δ + PCS1 complemented strain, on YPD with or without supplement, 3 d at 22°C:

• + 0.4 mM CdSO₄ — pcs1Δ showed greatest sensitivity (severe growth inhibition relative to WT and complemented strain; “greatest sensitivity in the deletion mutant was found for cadmium and copper”).
• + 10 mM CuSO₄ — pcs1Δ showed greatest sensitivity (severe growth inhibition relative to WT and complemented strain).
• + 2 mM ZnSO₄ — pcs1Δ showed a slight decrease in growth (also noted in text).
• Cobalt chloride, sodium arsenite, iron sulfate, manganese chloride, H₂O₂, and t-butyl-hydroperoxide (concentrations not specified beyond “toxic levels”) — no major difference between pcs1Δ and WT; data not shown in figure (text, p. 2 column 2 and p. 3 column 1).

PCS1 transcript regulation in Sporobolomyces (text, p. 3 column 1). Northern blot of PCS1 transcript in WT Sporobolomyces cultured overnight in YPD alone or with copper, cadmium, or zinc: no difference in transcript level with any of the three metal treatments versus YPD alone. The authors interpret this as evidence that PCS regulation in Sporobolomyces, as in many other species, is post-transcriptional (direct enzyme activation by the metal cation, consistent with the Grill et al. mechanism for plant PCS).

S. pombe complementation by Sporobolomyces PCS1 (Figure 3A). Ten-fold serial dilutions of S. pombe WT or pcs1Δ strains carrying empty pREP42, S. pombe PCS1 (pAS4), or Sporobolomyces PCS1 (pAS5), on YPD with or without 10 µM CdSO₄, 3 d at 30°C. The S. pombe pcs1Δ strain was Cd-sensitive (confirming prior reports). Cd sensitivity was complemented by either the S. pombe or the Sporobolomyces PCS1 copy — heterologous complementation works, indicating the Sporobolomyces protein is functionally equivalent to the S. pombe protein in Cd handling.

S. pombe pcs1 mutant Cu phenotypes (Figure 3B and text, p. 3 column 2). On YES + uracil (20 mg/L) + 0.625 mM CuSO₄, WT grew normally and was pigmented (brown), pcs1Δ showed slower growth and less pigmentation, indicating a Cu-homeostasis defect, not only Cu sensitivity. Strains MM72-4A (WT) and AS8 (pcs1Δ) were compared after 6 d at 22°C.

Pcs1-GFP mitochondrial localisation in S. pombe (Figure 4). Construct pAS6 (S. pombe PCS1–GFP fusion, expressed from pREP42 nmt promoter) transformed into the AS8 pcs1Δ strain → AS15. The fusion construct complemented Cd sensitivity (Figure 4A: 10-fold serial dilutions on YNB or YNB + 50 µM CdSO₄, 4 d at 22°C; WT + empty and pcs1Δ + PCS1-GFP grew at 50 µM Cd, pcs1Δ + empty did not), confirming the fusion is functional. GFP fluorescence appeared as punctate dots or filaments and co-localised with MitoTracker red staining (3 nM, 20 min, washed, re-suspended in PBS, examined on Olympus Fluoview FV300 confocal). Scale bar 10 µm.

PCS distribution across fungal genomes (Table 1, 375 genome sequences summed across nineteen lineage/sub-lineage rows). The rows are:

• Present: Chytridiomycota (P; 2 genomes; Catenaria, Spizellomyces); Neocallimastigomycota (P; 1; Piromyces); Blastocladiomycota (P; 2; Allomyces, Batrachochytrium); Glomeromycota (P; 1; Rhizophagus); Mucoromycotina (SP), Mucorales (O; 8; Mucor, Rhizopus, Phycomyces, Lichtheimia); Mucoromycotina, Mortierellales (O; 1; Mortierella); Kickxellomycotina (SP; 1; Coemansia); Ascomycota, Taphrinomycotina (SP; 7; Schizosaccharomyces, Saitoella); Ascomycota, Pezizomycotina (SP; 180; present only in class Pezizomycetes, absent in all other species — Ascobolus, Tuber, Wilcoxina, Pyronema); Basidiomycota, Pucciniomycotina (SP; 16; Sporobolomyces, Rhodotorula, Puccinia); Basidiomycota, Wallemiomycetes (SP; 2; Wallemia); Unclassified, Cryptomycota (1; Rozella); Unclassified, Monoblepharidomycetes (1; Gonapodya).
• Absent: Microsporidia (P; 5); Mucoromycotina, Entomophthoromycotina (SP; 1); Ascomycota, Saccharomycotina (SP; 35); Basidiomycota, Agaricomycotina (SP; 103); Basidiomycota, Ustilaginomycotina (SP; 8).
• Unknown: Mucoromycotina, Zoopagomycotina (SP; 0 sequenced).

Pcs1 protein features (text, p. 6). The C terminus is cysteine-rich in many homologs but positions do not align: S. pombe CCX₅CCX₃CC; Sporobolomyces CCX₈CXC; Phycomyces blakesleeanus homolog CX₂CX₃C; Catenaria anguillulae CCCX₁₁CXCC (X = any amino acid). Active-triad cysteine substitutions are present in four candidate PCS-like proteins from Mucoromycotina (form a 100 %-bootstrap clade in the phylogeny, Figure 5) — these may be enzymatically inactive or perform a different function.

Phylogeny support (Figure 5 and text, p. 7 column 1). Maximum-likelihood tree built on 243 conserved amino acids from PCS homologs, 100 bootstraps. Two PCS groups in Basidiomycota split by homologs from Ascomycota → possible horizontal gene transfer. Two PCS proteins in Catenaria anguillulae positioned in distinct parts of the phylogeny → possible interspecies exchange during its evolution. Puccinia graminis (Basidiomycota) has two homologs as a tandem gene duplication; most Mucoromycotina species have more than one copy (consistent with whole-genome / segmental duplications in those lineages, Ma et al. 2009; Schwartze et al. 2014).

GenBank deposition. Sporobolomyces sp. PCS1 genomic and cDNA sequence deposited under accession KJ000020 (text, p. 11 column 1).

Evidence Fitness

This source supports Context only for the wiki’s contamination-occurrence layer: no food, personal-care, soil, water, or human-exposure measurements are reported. Its evidentiary contribution is mechanistic and comparative — primary peer-reviewed laboratory work with appropriate internal controls (independent gene deletion in Sporobolomyces, complementation by the wild-type gene from both Sporobolomyces and S. pombe, heterologous expression in two ascomycete yeasts, reciprocal S. pombe complementation, GFP fluorescence co-localisation, 408-genome bioinformatic survey, 100-bootstrap phylogeny). It is appropriate for A-tier citation in the wiki when:

• cadmium describes fungal PCS-mediated Cd detoxification and the conservation of that mechanism across fungal lineages.
• copper discusses PCS contribution to essential-metal homeostasis (the Sporobolomyces pcs1Δ Cu-sensitivity result and the S. pombe pcs1Δ pigmentation result are the empirical anchors).
• zinc discusses fungal Zn handling and the modest but reproducible Zn growth defect in pcs1Δ.
• A future page on bioremediation, food-supply-chain mycology, or fungal-driven Cd mobilisation/immobilisation needs the conservation pattern (Table 1) to explain which fungal taxa can and cannot deploy PCS-based chelation.

It is not appropriate as evidence for any quantitative claim about Cd, Cu, Zn, or As concentrations in food, water, soil, or biological samples. The arsenic finding — that Sporobolomyces pcs1Δ shows no major sensitivity difference from WT on sodium arsenite — is a null result on yeast growth, not a measurement on any matrix, and should not be cited as occurrence data on arsenic-inorganic.

Methods anchors

• Sporobolomyces sp. strain IAM 13481 — wild type, leaf-surface isolate from a willow tree (Fay et al. 2004 cited as origin; reference 35 in paper).
• ura5 auxotroph AIS2 — spontaneous ura5 mutation in IAM 13481 (Kim H-S et al. 2009 cited as origin; reference 36).
• Gene replacement of PCS1 with URA5 selectable marker by homologous recombination; 1,334 / 1,232 bp flanks amplified with primers AS001-AS002 and AS003-AS004; URA5 amplified with ALID0562-ALID0564; three-fragment overlap PCR with AS001-AS004. Transformation into AIS2 by biolistic delivery on gold beads (Pitkin et al. 1996 method; reference 50). PCR confirmation (Figure 2B, primers AS005-AS006) and Southern blot confirmation (Figure 2C, BamHI / EcoRI / HindIII digests; AS020-AS004 probe; 32P-dCTP labeling). Complementation strain AS3 generated by reintroducing wild-type PCS1-URA5 fusion after recycling URA5 on 5-FOA.
• Sporobolomyces PCS1 cDNA cloning — reverse-transcribed from total RNA using oligo-dT primer + Superscript III; PCR with AS005, AS006 (genomic) or AS018, AS019; ligated into pYES2 (NdeI/XbaI) under GAL1 promoter for S. cerevisiae expression → pAS1, pAS2, pAS3; into pREP42 (BamHI/NdeI) under nmt promoter for S. pombe expression → pAS5.
• S. pombe PCS1 gene replacement with KanMX — 360 / 339 bp flanks on either side of KanMX cassette by overlap PCR (5′ flank with AS012, AS013; 3′ flank with AS014, AS015; KanMX with KanMX F / KanMX R). Transformation into MM72-4A by lithium acetate/PEG; selection on 50 or 100 µg/L G-418.
• S. pombe PCS1-GFP fusion (pAS6) — S. pombe PCS1 amplified with AS018, AS019, AS018-AS016 fragment; GFP with AISV066, ALID2091; overlap PCR with AS018-AISV066; cloned BamHI/NdeI into pREP42.
• Subcellular-localisation prediction software — PSORT II (Nakai & Horton 1999), MitoProt (Claros & Vincens 1996).
• Microscopy — Olympus Fluoview FV300 confocal. MitoTracker red (Invitrogen) at 3 nM, 20 min stain, PBS wash and re-suspension before imaging. Scale bar 10 µm in Figure 4B.
• Genome BLASTp — queries against MycoCosm (US DOE JGI), NCBI, and Broad Institute databases; queries closed 1 November 2014. ClustalW alignment; manual inspection for annotation errors; revised annotations generated in six cases (Pyronema confluens, Rhizophagus irregularis, Tuber melanosporum, Rhizopus oryzae, Batrachochytrium dendrobatidis, Phycomyces blakesleeanus).
• Phylogeny — MEGA6 (Tamura et al. 2013); best-fit ML model LG+G; all amino-acid sites; 100 bootstrap replicates; alignment 243 amino acids on the conserved core after trimming variable N- and C-terminal ends.

Verification notes

Source ingested fresh from raw/Manual Fetch Kimi /June 8/Kimi_Agent_Black Market Peptide Metal Survey/heavy_metals_peptides/39_Phytochelatin_Synthase_Is_Required_for_Tolerating_Metal_Toxi.pdf (SHA256 5cd9073a655914d7e9e2064377ed4b71512377a7b704260f6c7a8752f4dbbdde). Open-access CC-BY-4.0 BioMed Central article (DOI 10.1186/s40694-015-0013-3). All numeric values traced to the cited figure or text location on the indicated page of the published article (13 pages total). Strain table, primer table, and Table 1 lineage distribution transcribed in full. The paper reports null results for cobalt, sodium arsenite, iron sulfate, manganese chloride, H₂O₂, and t-butyl-hydroperoxide as growth-effect tests on Sporobolomyces pcs1Δ; these null findings are recorded in Key numbers but not propagated to frontmatter metals: [] beyond the four metals (Cd, Cu, Zn, iAs) for which the paper has substantive empirical content. No food matrix, ingredient, product, or jurisdiction is invoked, so the corresponding frontmatter arrays are empty by design — this is a mechanistic genetics/genomics paper and the routing layer should treat it as exposure-only/mechanistic context for the three metal pages noted in Evidence Fitness.

Audit subagent (2026-06-08) flagged two findings; both verified against the PDF and applied:

• ❌ Strain genotype mapping for AS9–AS14 was transposed. The initial ingest paired AS9 with AS12 (both as pcs1Δ + empty pREP42), AS10 with AS13, and AS11 with AS14, treating all six as pcs1Δ-background strains. Verified against Table 2 (p. 9, Parent/origin column): AS9, AS10, AS11 carry the MM72-4A (WT ura4-D18) background and are the wild-type-background control panel; AS12, AS13, AS14 carry the AS8 (pcs1Δ) background and are the complementation-assay panel; AS15 (pcs1Δ + PCS1-GFP) is correctly attributed in both versions. Corrected the strain-table paragraph in Key numbers to separate the two background trios. Figure 4A’s strain triplet (AS09 / AS12 / AS15) is independently consistent with the corrected mapping.
• ❌ “408 fungal genome sequences” was incorrect. The initial ingest stated 408 genomes examined; the number does not appear in Table 1 (p. 6), Methods, or Results body. Independent sum of Table 1’s “Genome sequences examined” column: 2+1+2+5+1+8+1+1+0+1+7+35+180+103+16+8+2+1+1 = 375. Corrected to 375 in the lead paragraph, the third bullet of Why this matters, the Table 1 distribution paragraph heading, and the present/absent narrative.

No other audit findings; the Key numbers Cd unit-conversion (1 µM CdSO₄ ≈ 112 µg Cd/L), C-terminal cysteine motifs, slug vocabulary, methods anchors, brand firewall, and HMTc firewall all passed independent re-verification.

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