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RESEARCH ARTICLE Disruption of a cystine transporter downregulates expression of genes involved in sulfur regulation and cellular respiration Jessica A. Simpkins1, Kirby E. Rickel1, Marianna Madeo2, Bethany A. Ahlers1, Gabriel B. Carlisle1, Heidi J. Nelson1, Andrew L. Cardillo2, Emily A. Weber1, Peter F. Vitiello2, David A. Pearce2 and Seasson P. Vitiello1,* 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. KEY WORDS: Cystine, ERS1, CTNS 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). Without Cystinosin, the transport of cystine out of the lysosome is severely restricted, leading to cystine accumulation. Cystine is naturally found in the lysosome as result of protein hydrolysis and the influx of extracellular cystine (Danpure et al., 1986; Thoene and Lemons, 1980). Cystinosin exports cystine from the lysosome to the cytosol, where it can be reduced to cysteine to be used in downstream processes (Kalatzis et al., 2001). The absence of Cystinosin results in a surplus of cystine in the lysosome and eventual apoptosis (Jonas et al., 1982; Park et al., 2002; Schulman et al., 1969). Direct lysosomal dysfunction may contribute to cell death, but more likely, a lack of cystine recycling weakens the cell. For example, cysteine is the limiting precursor in glutathione synthesis, a tripeptide that functions in the elimination of oxidants that can damage DNA, proteins, and lipids. It is possible that apoptosis occurs secondarily to cystine storage, triggered by rampant reactive oxygen species that damage cellular components at higher rates due to a shortage of cysteine needed to synthesize sufficient levels of glutathione. In fact, 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). However, depleted ATP levels may also contribute to apoptosis (Coor et al., 1991; Kumar and Bachhawat, 2010; Levtchenko et al., 2006; Wilmer et al., 2008). There is also evidence that the lysosomes fragment and release cystine in mass into the cytosol, where the cystine is quickly reduced to cysteine. The large quantities of free cysteine then cysteinylate proapoptotic proteins, such as PKCδ (Park et al., 2002, 2006; Park and Thoene, 2005; Thoene, 2007). In addition, 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. The amino acid sequence of Cystinosin is 43% identical and 64% similar over 102 amino acids to a transmembrane protein encoded by ERS1 in Saccharomyces cerevisiae (Town et al., 1998). The encoded yeast protein, Ers1p, localizes to the vacuole, an organelle analogous to the lysosome in mammalian cells (Gao et al., 2005). ERS1 was originally identified as a high-copy suppressor of erd1-Δ, with ERD1 encoding a protein necessary for ER protein retention, although the exact relationship between ERS1 and ERD1 remains unknown (Hardwick et al., 1990; Hardwick and Pelham, 1990). Deletion of ERS1, ers1-Δ, is lethal in the presence of the antibiotic hygromycin B in a strain-dependent manner. Human CTNS driven by the putative ERS1 promoter complements ers1-Δ when the cells are grown in the presence of hygromycin B, indicating that proteins Ers1p and Cystinosin share common functions (Gao et al., 2005). The hygromycin B sensitivity can be suppressed by overexpression of MEH1, which encodes a protein involved in vacuolar acidification and general amino acid permease (Gap1p) localization (Gao and Kaiser, 2006; Gao et al., 2005). Ers1p has been proposed to be a cystine transporter through complementation studies using CTNS, but Ers1p-dependent transport has not previously been reported (Gao et al., 2005). In this study, we performed a biochemical transport assay to confirm the existence of an Ers1p-dependent cystine transport system. Received 9 February 2016; Accepted 13 April 2016 Because human cells lacking Cystinosin accumulate 100-fold more 1Biology Department, Augustana University, Sioux Falls, SD, USA 57197. 2Sanford Research Children’s Health Research Center, Sioux Falls, SD, USA 57104. *Author for correspondence (seasson.vitiello@augie.edu) S.P.V., 0000-0001-5817-4685 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 689 © 2016. Published by The Company of Biologists Ltd | Biology Open (2016) 5, 689-697 doi:10.1242/bio.017517 Biology Open
Object Description
Title | Disruption of a cystine transporter downregulates expression of genes involved in sulfur regulation and cellular respiration |
Author(s) | Simpkins, Jessica A.; Rickel, Kirby E.; Madeo, Marianna; Ahlers, Bethany A.; Carlisle, Gabriel B.; Nelson, Heidi J.; Cardillo, Andrew L.; Webers, Emily A.; Vitiello, Peter F.; Pearce, David A.; Vitiello, Seasson P. |
Augustana Author(s) | Vitiello, Seasson P. |
Department | Biology |
Subject (LC) |
Cystinosis Gene expression |
Year | 2016 |
Journal Title | Biology Open |
Publishing agency | Company of Biologists |
Type | Journal Article |
Medium | Text |
Format - Digital | |
Link | https://dx.doi.org/10.1242/bio.017517 |
Language | English |
Rights | Green |
Collection | Augustana Faculty Authors |
Description
Title | Page 1 |
Abstract | RESEARCH ARTICLE Disruption of a cystine transporter downregulates expression of genes involved in sulfur regulation and cellular respiration Jessica A. Simpkins1, Kirby E. Rickel1, Marianna Madeo2, Bethany A. Ahlers1, Gabriel B. Carlisle1, Heidi J. Nelson1, Andrew L. Cardillo2, Emily A. Weber1, Peter F. Vitiello2, David A. Pearce2 and Seasson P. Vitiello1,* 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. KEY WORDS: Cystine, ERS1, CTNS 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). Without Cystinosin, the transport of cystine out of the lysosome is severely restricted, leading to cystine accumulation. Cystine is naturally found in the lysosome as result of protein hydrolysis and the influx of extracellular cystine (Danpure et al., 1986; Thoene and Lemons, 1980). Cystinosin exports cystine from the lysosome to the cytosol, where it can be reduced to cysteine to be used in downstream processes (Kalatzis et al., 2001). The absence of Cystinosin results in a surplus of cystine in the lysosome and eventual apoptosis (Jonas et al., 1982; Park et al., 2002; Schulman et al., 1969). Direct lysosomal dysfunction may contribute to cell death, but more likely, a lack of cystine recycling weakens the cell. For example, cysteine is the limiting precursor in glutathione synthesis, a tripeptide that functions in the elimination of oxidants that can damage DNA, proteins, and lipids. It is possible that apoptosis occurs secondarily to cystine storage, triggered by rampant reactive oxygen species that damage cellular components at higher rates due to a shortage of cysteine needed to synthesize sufficient levels of glutathione. In fact, 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). However, depleted ATP levels may also contribute to apoptosis (Coor et al., 1991; Kumar and Bachhawat, 2010; Levtchenko et al., 2006; Wilmer et al., 2008). There is also evidence that the lysosomes fragment and release cystine in mass into the cytosol, where the cystine is quickly reduced to cysteine. The large quantities of free cysteine then cysteinylate proapoptotic proteins, such as PKCδ (Park et al., 2002, 2006; Park and Thoene, 2005; Thoene, 2007). In addition, 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. The amino acid sequence of Cystinosin is 43% identical and 64% similar over 102 amino acids to a transmembrane protein encoded by ERS1 in Saccharomyces cerevisiae (Town et al., 1998). The encoded yeast protein, Ers1p, localizes to the vacuole, an organelle analogous to the lysosome in mammalian cells (Gao et al., 2005). ERS1 was originally identified as a high-copy suppressor of erd1-Δ, with ERD1 encoding a protein necessary for ER protein retention, although the exact relationship between ERS1 and ERD1 remains unknown (Hardwick et al., 1990; Hardwick and Pelham, 1990). Deletion of ERS1, ers1-Δ, is lethal in the presence of the antibiotic hygromycin B in a strain-dependent manner. Human CTNS driven by the putative ERS1 promoter complements ers1-Δ when the cells are grown in the presence of hygromycin B, indicating that proteins Ers1p and Cystinosin share common functions (Gao et al., 2005). The hygromycin B sensitivity can be suppressed by overexpression of MEH1, which encodes a protein involved in vacuolar acidification and general amino acid permease (Gap1p) localization (Gao and Kaiser, 2006; Gao et al., 2005). Ers1p has been proposed to be a cystine transporter through complementation studies using CTNS, but Ers1p-dependent transport has not previously been reported (Gao et al., 2005). In this study, we performed a biochemical transport assay to confirm the existence of an Ers1p-dependent cystine transport system. Received 9 February 2016; Accepted 13 April 2016 Because human cells lacking Cystinosin accumulate 100-fold more 1Biology Department, Augustana University, Sioux Falls, SD, USA 57197. 2Sanford Research Children’s Health Research Center, Sioux Falls, SD, USA 57104. *Author for correspondence (seasson.vitiello@augie.edu) S.P.V., 0000-0001-5817-4685 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 689 © 2016. Published by The Company of Biologists Ltd | Biology Open (2016) 5, 689-697 doi:10.1242/bio.017517 Biology Open |