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
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© 2016. Published by The Company of Biologists Ltd | Biology Open (2016) 5, 689-697 doi:10.1242/bio.017517
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