Yct1p, a novel, high-affinity, cysteine-specific transporter from the yeast Saccharomyces cerevisiae

Abstract

Cysteine transport in the yeast Saccharomyces cerevisiae is mediated by at least eight different permeases, none of which are specific for cysteine. We describe a novel, high-affinity, (K(m) = 55 microM), cysteine-specific transporter encoded by the ORF YLL055w that was initially identified by a combined strategy of data mining, bioinformatics, and genetic analysis. Null mutants of YLL055w, but not of the other genes encoding for transporters that mediate cysteine uptake such as GAP1, GNP1, MUP1, or AGP1 in a met15Delta background, resulted in a growth defect when cysteine, at low concentrations, was provided as the sole sulfur source. Transport experiments further revealed that Yll055wp was the major contributor to cysteine transport under these conditions. The contributions of the other transporters became relevant only at higher concentrations of cysteine or when YLL055w was either deleted or repressed. YLL055w expression was repressed by organic sulfur sources and was mediated by the Met4p-dependent sulfur regulatory network. The results reveal that YLL055w encodes the principal cysteine transporter in S. cerevisiae, which we have named YCT1 (yeast cysteine transporter). Interestingly, Yct1p belongs to the Dal5p family of transporters rather than the amino acid permease family to which all the known amino acid transporters belong.

Figure 1.—

YLL055w disruption in the met15Δ background results in a growth defect at low cysteine concentrations. The met15Δ, met15Δ yll055wΔ, met15Δ yil166cΔ, and met15Δ yal067cΔ strains were grown to exponential phase in minimal ammonia medium containing methionine, harvested, washed, and resuspended in water and serially diluted to give 0.2, 0.02, 0.002, and 0.0002 OD₆₀₀ of cells. A total of 10 μl of these dilutions was spotted on minimal ammonia medium containing different concentrations of cysteine (Cys) and dl-homocysteine (dl-Hcys). The photographs were taken after 3 days of incubation at 30°.

Figure 2.—

The YLL055w ORF expressed from TEF promoter complements the growth defect observed in met15Δ yll055wΔ at low cysteine concentrations. Plasmid-bearing YLL055w under the TEF and the corresponding vector were transformed into met15Δ and met15Δ yll055wΔ (yll055wΔ). The dilution spotting was done as described in materials and methods and in the legend to Figure 1.

Figure 3.—

YLL055w disruption in met15Δ and met15Δ gnp1Δ mup1Δ results in decreased uptake of [³⁵S]cysteine, which is restored by the YLL055w ORF expressed from TEF promoter. Strains—met15Δ, met15Δ yll055wΔ, met15Δ gnp1Δ mup1Δ, met15Δ gnp1Δ mup1Δ yll055wΔ, and met15Δ yll055wΔ complemented with YLL055w under the TEF promoter and the corresponding vector—were grown in minimal ammonia medium containing 2 μm methionine and used for the transport assay as described in materials and methods. Log-phase cells were incubated with 50 μm [³⁵S]cysteine for different time intervals and counts were taken to determine the intracellular cysteine accumulation with time. Data are shown as mean ± SD (n = 3).

Figure 4.—

Effect of YLL055w disruption on utilization of cysteine as the sole sulfur source in different strain backgrounds deleted in the major cysteine permeases in the presence of different nitrogen sources. (A) Strains—met15Δ, met15Δ yll055wΔ, met15Δ mup1Δ, met15Δ mup1Δ yll055wΔ, met15Δ agp1Δ, met15Δ agp1Δ yll055wΔ, met15Δ gap1Δ, met15Δ gap1Δ yll055wΔ, met15Δ gnp1Δ, and met15Δ gnp1Δ yll055wΔ—were used for the dilution spotting on ammonia as the nitrogen source in minimal media. (B) Strains—met15Δ, met15Δ yll055wΔ, met15Δ gap1Δ, met15Δ gap1Δ yll055wΔ, met15Δ agp1Δ, and met15Δ agp1Δ yll055wΔ were analyzed for growth on minimal proline medium by dilution spotting.

Figure 5.—

Lineweaver–Burke plot for cysteine uptake in the met15Δ strain to determine kinetic parameters of Yct1p. The initial rate of cysteine uptake was measured at cysteine concentrations ranging from 0.01 to 0.1 mm by harvesting cells incubated with radiolabeled cysteine at 30- and 90-sec time intervals. Data are representative of three experiments (done in duplicates).

Figure 6.—

Expression of YLL055w is under strong sulfur regulation in both met15Δ (ABC 733) and MET15 (ABC 734) strains. (A) β-Galactosidase reporter assay–plasmid pYLL055w-600, bearing 600-bp upstream sequences of YLL055w fused upstream of the LacZ ORF, was transformed into met15Δ and MET15 strains and the transformants were grown in the presence of different sulfur sources and assayed for β-galactosidase enzyme activity as described in materials and methods. Data are shown as mean ± SD (n = 5). (B) Cysteine uptake. Strains met15Δ and met15Δ yll055wΔ were grown in minimal ammonia medium containing 2 μm methionine (nonrepressive sulfur) and 200 μm methionine (repressive sulfur) and the initial rate of [³⁵S]cysteine uptake was calculated by measuring the radiolabeled cysteine accumulated in the cells at 30- and 90-sec time intervals. Wild-type (MET15) and yll055wΔ strains were also grown in minimal ammonia medium and the initial rate of cysteine uptake was determined. Data are shown as mean ± SD (n = 3).

Figure 7.—

Multiple sequence alignment of the YLL055w upstream sequences of different Saccharomyces species to identify conserved sequence stretches. The YLL055w upstream activating sequences of S. cerevisiae, S. bayanus, S. mikatae, S. paradoxus, and S. kudriavzevii were extracted and multiple sequence alignment was carried out using the webserver (http://203.90.127.21/anand/sacch_prom_pat.html; Kohli et al. 2004). A sequence alignment stretch corresponding to the −396 to −157 upstream region of YLL055w from S. cerevisiae is shown. The conserved sequence stretch at −385, resembling the sulfur regulatory motif II-AAANTGTGG, is boxed.

Figure 8.—

β-Galactosidase reporter assay of different deletion constructs of the YLL055w promoter to identify the functionally important cis motifs in it. Multicopy plasmids bearing different upstream sequences fused to the LacZ ORF were transformed into the met15Δ strain and β-galactosidase enzyme activity was determined after growing cells in a poor sulfur source (2 μm methionine) and in a rich sulfur source (250 μm cysteine).