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

Jessica A Simpkins,David A Pearce,Andrew L Cardillo,Emily A Weber,Seasson P Vitiello,Peter F Vitiello,Gabriel B Carlisle,Bethany A Ahlers,Heidi J Nelson,Kirby E Rickel,Marianna Madeo

doi:10.1242/bio.017517

Jessica A Simpkins, David A Pearce + Show 9 more

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https://doi.org/10.1242/bio.017517

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Journal: Biology open	Publication Date: May 3, 2016
Citations: 41	License type: CC BY 3.0

Affiliation: Augustana University, Sanford Research

Abstract

ABSTRACTCystine 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.

Highlights

Loss-of-function mutations in mammalian CTNS result in the absence of a cystine effluxer, Cystinosin (Gahl et al, 2002; Kalatzis et al, 2001; Town et al, 1998)
That ers1-Δ cells do not accumulate a significantly higher abundance of intracellular cystine compared to the ERS1+ parental cells, and they show no difference in growth and survival
Ers1p-dependent cystine transport While previous studies have supported that ERS1 and CTNS are orthologous, it had not been biochemically demonstrated that Ers1p transports cystine

Summary

Introduction

Loss-of-function mutations in mammalian CTNS result in the absence of a cystine (oxidized dicysteine) effluxer, Cystinosin (Gahl et al, 2002; Kalatzis et al, 2001; Town et al, 1998). Cystinosin exports cystine from the lysosome to the cytosol, where it can be reduced to cysteine to be used in downstream. Cysteine is the limiting precursor in glutathione synthesis, a tripeptide that functions in the elimination of oxidants that can damage DNA, proteins, and lipids. Lower levels of glutathione have been observed in cells lacking Cystinosin (Chol et al, 2004; Laube et al, 2006; Levtchenko et al, 2006; Mannucci et al, 2006). Cystine accumulation may be affecting the cell in using a yet-uncharacterized mechanism These mechanisms may not be mutually exclusive, and it is likely that a combination of these mechanisms is responsible for the observed increase in the rate of apoptosis in cells lacking Cystinosin

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