Genetic alterations in the TRiC complex affect arsenic susceptibility. (A) As(III)-sensitive heterozygous diploid YKOs identified by genome-wide mutant fitness profiling. Essential genes are colored in red and nonessential genes in black. The network diagram was created with Cytoscape 2.0 (Shannon et al. 2003). (B) Sensitivity of the heterozygous diploid YKOs of all eight TRiC subunits to As(III) at 450 μm. An ho/hoΔ mutant phenotypically indistinguishable from a wild-type yeast served as the control strain. (C) An hoΔ mutant harboring a vector or a plasmid overexpressing a TRiC subunit as indicated was tested for growth on solid SC −Ura that either contained or lacked 1 mm As(III).

The rate of protein synthesis and free β-tubulin modulate yeast arsenic sensitivity. (A) The distribution of arsenic-resistant haploid YKOs according to biological processes affected. This plot was derived from Table S3. (B) Cells of indicated genotypes were grown in an SC medium that either contained or lacked 800 μm of As(III). Cycloheximide was used at 10 ng/ml. (C) One of the α-tubulin genes TUB3 was overexpressed from a 2μ plasmid in mutants defective in microtubule biogenesis. Cells were grown on solid SC −Ura either in the presence or absence of As(III). As(III) concentration used was 600 μm for the hoΔ, tub3Δ, and cin2Δ mutants and 50 μm for the gim3Δ and pfd1Δ mutants. (D) The β-tubulin gene TUB2 and the actin gene ACT1 were expressed in mutants of indicated genotypes from a centromere-based plasmid. Cells were grown on solid SC that lacked uracil (SC −Ura) either in the presence or absence of 200 μm of As(III). (E) Strains of indicated genotypes were grown on solid YPD with or without As(III) (1 mm) or benomyl (20 μg/ml).

A genome-wide screen identifies double mutants hypersensitive to low concentrations of arsenic. (A) A high-efficiency pfd1Δ∷URA3 gene disruption cassette was transformed into a pool of haploid-convertible heterozygous diploid yeast knockout mutants. After sporulation, a pool of haploid double knockout mutants were derived from the heterozygous diploid double mutant pool in the presence of 5 μm of As(III). A separate pool of haploid single and double mutants were also similarly derived either in the presence or absence of 5 μm of As(III). Relative representation of each knockout mutation in both pools was compared by TAG-array analysis (Pan et al. 2004). (B) Haploid-convertible ARR1/arr1Δ∷kanMX PFD1/pfd1Δ∷URA3 and ARR3/arr3Δ∷kanMX PFD1/pfd1Δ∷URA3 diploid double mutants were sporulated and spotted on a haploid selection medium that either lacked both uracil and G418 (to select for Ura⁺ single and double mutants), contained both uracil and G418 (to select for G418-resistant single and double mutants), or lacked uracil but contained G418 (to select for the double mutants). Cell growth under each condition was assessed both in the absence (control) and presence of 2 μm or 5 μm of As(III) and photographed.

Arsenic-sensitive haploid YKOs and their genetic relationships with CCT1. (A) As(III)-sensitive haploid deletion mutants identified by genome-wide mutant fitness profiling. A total of 191 were individually verified to be sensitive to As(III) at 400 μm. The number of mutants in each biological process affected and the corresponding percentage among all mutations identified were listed. This plot was derived from Table S2. (B) Growth of representative arsenic-sensitive haploid YKOs of indicated genotypes on a solid synthetic complete (SC) medium that either lacked or contained As(III) at 40 μm, 150 μm, and 800 μm. (C) Partial suppression of the arsenic sensitivity by CCT1 overexpression in various mutants.

As(III) inhibits TRiC functions. (A) Wild-type yeast (BY4743) cells were grown in liquid YPD either in the absence or presence of 1 mm of As(III) for 3 hr. Actin was stained with rhodamine-phalloidin and microtubules were visualized with an anti-α–tubulin antibody. (B) ATP-depleted cell lysates prepared from yeast cells expressing Cdc55–3HA grown in liquid YPD that either contained or lacked 1 mm As(III) were subjected to immunoprecipitation with anti-HA antibody. Western blots of the total cell extracts (5μg) and the immunoprecipitates (IP) from lysates containing 200 μg total protein were analyzed with an anti-HA antibody and an antibody against one of the TRiC subunits Cct5. (C) In vitro binding and folding of denatured [³⁵S]-actin by bovine TRiC both in the presence and absence of 1 mm As(III) were assessed by native gel analysis followed by autoradiography. Three (2, 3, and 4) different schemes of arsenic treatment were tested. Native [³⁵S]-actin samples incubated with or without As(III) were included as controls (right two lanes). The extent of As(III) inhibition in each condition was quantified for three independent experiments and expressed as percentage of actin folding relative to the untreated control. (D) In vitro rhodanese folding by the M. maripaludis TRiC-like chaperonin (Mm-Cpn) was assessed in the presence and absence of 1 mm sodium arsenite as described (Kusmierczyk and Martin 2003).