Disruption of a cystine transporter downregulates expression of genes involved in sulfur regulation and cellular respiration

Abstract

Cystine and cysteine are important molecules for pathways such as redox signaling and regulation, and thus identifying cellular deficits upon deletion of the Saccharomyces cerevisiae cystine transporter Ers1p allows for a further understanding of cystine homeostasis. Previous complementation studies using the human ortholog suggest yeast Ers1p is a cystine transporter. Human CTNS encodes the protein Cystinosin, a cystine transporter that is embedded in the lysosomal membrane and facilitates the export of cystine from the lysosome. When CTNS is mutated, cystine transport is disrupted, leading to cystine accumulation, the diagnostic hallmark of the lysosomal storage disorder cystinosis. Here, we provide biochemical evidence for Ers1p-dependent cystine transport. However, the accumulation of intracellular cystine is not observed when the ERS1 gene is deleted from ers1-Δ yeast, supporting the existence of modifier genes that provide a mechanism in ers1-Δ yeast that prevents or corrects cystine accumulation. Upon comparison of the transcriptomes of isogenic ERS1+ and ers1-Δ strains of S. cerevisiae by DNA microarray followed by targeted qPCR, sixteen genes were identified as being differentially expressed between the two genotypes. Genes that encode proteins functioning in sulfur regulation, cellular respiration, and general transport were enriched in our screen, demonstrating pleiotropic effects of ers1-Δ. These results give insight into yeast cystine regulation and the multiple, seemingly distal, pathways that involve proper cystine recycling.

Keywords: CTNS; Cystine; ERS1.

Fig. 1.

Ers1p-dependent cystine transport. (A) Western blot, representative of two replicates, showing that upon overexpression, Ers1p-V5 localizes to both the vacuole (Vac) and plasma membrane (PM). ERS1-V5 was overexpressed using a galactose inducible promoter. Plasma membrane and vacuoles were enriched from whole cell lysate (WCL). Western blotting using an anti-V5 antibody indicated that Ers1p-V5 was located both in vacuoles (as determined by vacuolar marker Vph1p) and plasma membrane (as determined by plasma membrane marker Pma1p). The expected size of Ers1p-V5 was 30 kDa, with a larger band located at approximately 50 kDa. Dpm1p is an ER marker as an indicator of ER contamination. (B) Transport was measured by resuspending cells overexpressing either ERS1 or CTNS in buffer at pH 6 and measuring the intracellular cystine over time. (C) Graph of Ers1p-dependent transport of cystine. Cells overexpressing ERS1 or CTNS showed increased intracellular cystine over time, while cells with vector alone did not (mean±s.e.m.; n=4 cultures). (D) ERS1 expression in can1-Δ did not rescue arginine transport, whereas CAN1 expression did (mean±s.e.m.; n=4 cultures).

Fig. 2.

Genetic interactions with ERS1. Genes found to have differential expression in ers1-Δ were categorized into several functional groups, with energy and sulfur homeostasis as the two major groups. Most genes fell into more than one group.

Fig. 3.

Cells lacking ERS1 have increased aerobic respiration in vivo. Cells were grown to mid-log phase in minimal media and shifted to minimal media with either 2% glucose or 2% lactate. (A) Aerobic respiration is increased in media containing lactate, a non-fermentable carbon source, but not in glucose as per measurements using an FX Flux Analyzer. (B) Extracellular acidification was the same in the two genotypes in both carbon sources. Data represented as mean±s.e.m.; n=3 cultures. Statistical comparisons were made using Student's t-test (*P<0.05).

Fig. 4.

ers1-Δ cells have decreased survival in menadione and wild-type glutathione levels. (A) The amount of glutathione (GSH) in cells grown to mid-log phase in minimal media was quantified (mean±s.e.m.; n=3 cultures). Since GSH2 encodes the protein that catalyzing a major step in the glutathione pathway (Inoue et al., 1998), gsh2-Δ served as a control. (B) Cells (n=3 cultures) were incubated in 100 μM menadione or vehicle for 4 h, and then plated on rich media at 30°C. After 2 days, colonies were counted. Drug treatment was normalized to vehicle controls to calculate percent survival. Superoxide dismutase is encoded by SOD1. The deletion strain, which will not survive in menadione, was used as a control. (C) Graph of survival rates in B (mean±s.e.m.). Statistical comparisons were made using two-way ANOVA and Bonferroni post-test (*P<0.001).