Coordinate Control of Sphingolipid Biosynthesis and Multidrug Resistance in Saccharomyces cerevisiae

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Saccharomyces cerevisiae cells lacking their mitochondrial DNA ( 0 cells) respond to this loss of genetic information by induction of a program of nuclear gene expression called the retrograde response. Expression of genes involved in multidrug resistance and sphingolipid biosynthesis is coordinately induced in 0 cells by the zinc cluster transcription factor Pdr3p. In this report, we identify a membrane protein involved in control of intracellular levels of a sphingolipid precursor as a transcriptional target of the Pdr3p-mediated retrograde response. These sphingolipid precursors are called long chain bases (LCBs) and increased LCB levels are growth inhibitory. This membrane protein has been designated Rsb1p and has previously been shown to act as a LCB transporter protein and to be a component of the endoplasmic reticulum. These earlier studies used an amino-terminal truncated form of Rsb1p. Here we employ a full-length form of Rsb1p and find that this protein is localized to the plasma membrane and is modified by N-linked glycosylation. Two glycosylation sites are present in the Rsb1p and both are required for normal LCB resistance. Mutational analysis of the RSB1 promoter revealed that two Pdr3p binding sites are present and both of these are required for normal retrograde induction of transcription. LCB tolerance is strongly increased in 0 cells but this increase is ablated in 0 rsb1⌬ cells. Together, these data indicate Pdr3p activation of RSB1 transcription is an important feature of the retrograde response allowing normal detoxification of an endogenous sphingolipid precursor.Sphingolipids are important lipid constituents of eukaryotic membranes. Key intermediates in the biosynthesis of sphingolipids are the long chain bases (LCBs).2 In Saccharomyces cerevisiae, these LCBs are dihydrosphingosine and phytosphingosine (PHS) (reviewed in Ref. 1). These LCBs are utilized by ceramide synthase to form ceramide that is a well known bioactive lipid (reviewed in Refs. 2 and 3). However, LCBs and their phosphorylated forms (LCBPs) have also recently been found to serve as potent signaling molecules. LCBs appear to act as inhibitors of proliferation, whereas increased LCBP levels stimulate growth (recently reviewed in Ref. 4). Control of the biological levels of these signaling lipids is an essential feature for ensuring both normal lipid composition of membranes and proper metabolic regulation.The central importance of these signaling and structural lipids is likely to explain the multitude of regulatory mechanisms modulating their levels. These include enzymes like ceramidases that break down ceramide (5, 6) and the LCBP lyase that cleaves these phosphorylated lipids into ethanolamine and a long chain aldehyde (7). More recent experiments in the yeast S. cerevisiae have identified a new membrane protein that is thought to efflux LCBs out of the cell. This protein has been designated Rsb1p (resistant to sphingoid bases) as it was identified as a high-copy suppressor of the PHS hypersensitivity of a dpl1⌬ strain (...

Section: Discussionmentioning

confidence: 77%

mentioning

confidence: 96%

Long Chain Base Tolerance in Saccharomyces cerevisiae Is Induced by Retrograde Signals from the Mitochondria

Panwar

Moye‐Rowley

2006

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“…In summary, our studies strongly suggest that Rdr1p acts through Pdr1p/Pdr3p binding sites. However, many genes (11,(13)(14)(15)(16)(17) (like SNQ2) that have been shown to contain PDREs and to be regulated by Pdr1p/ Pdr3p are not affected by removal of Rdr1p. Thus, the effect of Rdr1p appears to be mediated by a subset of the target genes of FIG.…”

Section: Discussionmentioning

confidence: 99%

Zinc Cluster Protein Rdr1p Is a Transcriptional Repressor of the PDR5 Gene Encoding a Multidrug Transporter

Hellauer¹,

Akache²,

MacPherson³

et al. 2002

The yeast PDR5 gene encodes an efflux pump that confers multidrug resistance. Expression of PDR5 is positively regulated by the transcription factors Pdr1p and Pdr3p that recognize the same pleiotropic drug resistance elements (PDREs) in the PDR5 promoter. Pdr1p and Pdr3p belong to the Gal4p family of zinc cluster proteins. The function of RDR1 (YOR380W), which also encodes a member of this family, is unknown. To identify target genes for Rdr1p, we have performed wholegenome analysis of gene expression with DNA microarrays. Our results show that Rdr1p is a transcriptional repressor of five genes, including PDR5. A ⌬rdr1 strain has increased resistance to cycloheximide, as expected from the overexpression of PDR5. In addition, the activity of a PDR5-lacZ reporter is increased in a ⌬rdr1 strain. All (but one) genes affected by removal of Rdr1p contain PDREs in their promoters. We tested if the effect of Rdr1p is mediated through PDREs by inserting this DNA element in front of a minimal promoter. Activity of this reporter was increased in a ⌬rdr1 strain. Moreover, mutations known to reduce binding of Pdr1/ Pdr3p abolished the induction observed in the ⌬rdr1 strain. Thus, we have identified a transcriptional repressor involved in the control of multidrug resistance.Multidrug resistance is a widespread phenomenon that allows organisms ranging from bacteria to humans to defend themselves against a variety of toxic compounds. Drug resistance is mediated through membrane-bound transporters that act as drug efflux pumps. This process has been extensively studied in the yeast Saccharomyces cerevisiae. Two major classes of multidrug transporters have been identified: the major facilitator superfamily (MFS) 1 (1) and the ABC (ATP binding cassette) family of transporters (2-4). ABC transporters act in an ATP-dependent manner, and their overexpression leads to increased drug resistance. Members of the family of ABC transporters include Pdr5p, Snq2p, and Yor1p (2).The transcriptional activators Pdr1p and Pdr3p control the expression of many ABC transporters (3, 4). These activators belong to a family of transcriptional regulators called zinc cluster or binuclear cluster proteins (5-7). Members of this family contain six highly conserved cysteines that coordinate binding to two zinc atoms to allow proper folding of the DNA binding domain. The cysteine-rich region (or zinc finger) is usually followed by a short linker sequence that bridges the zinc finger to a dimerization domain (6). This domain is involved in recognition of specific DNA sequences through interaction of the zinc finger with DNA (for references see Ref. 8). Pdr1p and Pdr3p both recognize CGG triplets oriented in opposite directions (CCGCGG) to form an everted repeat (9). This motif is found in the target genes of Pdr1p and Pdr3p (10 -17). For example, three binding sites called PDREs (pleiotropic drug response elements) for Pdr1p and Pdr3p have been mapped in the PDR5 promoter (10, 12). Thus, Pdr1p and Pdr3p recognize the same DNA sequences in target promoters f...

“…About 20 g of total RNA were separated on a 1.4% agarose gel and transferred to nylon membranes (Amersham Biosciences). Northern blots were hybridized with PCR-amplified probes and radiolabeled by incorporation of [␣- 32 P]dCTP and a MegaPrime labeling kit (Amersham Biosciences). Methylene blue staining was used as control for equal RNA loading.…”

Section: Methodsmentioning

confidence: 99%

Weak Organic Acids Trigger Conformational Changes of the Yeast Transcription Factor War1 in Vivo to Elicit Stress Adaptation

Gregori

Schüller

Frohner

et al. 2008

The Saccharomyces cerevisiae zinc cluster regulator War1 mediates an essential transcriptional and adaptive response to weak organic acid stress. Here we investigate the mechanism of War1 activation upon weak acid stress. We identified several gain-of-function WAR1 alleles mapping to the central War1 region. These mutations constitutively increase levels of the plasma membrane ABC transporter Pdr12, the main War1 target mediating stress adaptation. Functional analysis of War1 reveals that the central region and its C-terminal activation domain are required for function. Notably, the native DNAbinding and dimerization domains appear dispensable for War1 activity, because they can be replaced by a LexA DNA-binding domain. Chromatin immunoprecipitation demonstrates elevated promoter affinity of activated War1, because its PDR12 promoter association increases upon stress. Hyperactive WAR1 alleles have constitutively high PDR12 promoter association. Furthermore, fluorescence resonance energy transfer of functional CFP-War1-YFP proteins also demonstrates conformational changes of stress-activated War1 in vivo. Our results suggest a mechanism whereby War1 activation is accompanied by conformational changes enhancing promoter association, thus initiating the adaptation process.Weak organic acids, such as potassium sorbate, calcium propionate, and sodium benzoate, are commonly used to preserve foods and beverages (1). In Saccharomyces cerevisiae, uncharged weak acids diffuse across the plasma membrane and dissociate inside the cell, causing intracellular acidification, free radical production, and oxidative stress (1), as well as inhibition of several metabolic processes (2). The yeast ABC transporter Pdr12 confers resistance to monocarboxylic acids with chain lengths up to C 7 (3, 4). Only organic acids but not other stresses cause a dramatic induction of PDR12 transcription and a concomitant increase of Pdr12 (4, 5). A physiological role of Pdr12 is suggested from its proposed ability to extrude amino acid catabolism by-products (6). Weak acid regulation of PDR12 occurs at the level of transcription and requires the Zn(II) 2 Cys 6 zinc cluster transcription factor War1, which recognizes and binds response elements within the PDR12 promoter (7). War1 is functionally conserved among several yeast species, including fungal pathogens. An orthologue mediating sorbate tolerance was identified in the distantly related human fungal pathogen Candida albicans (8), but the mechanism by which weak organic acids cause War1 activation is currently an open question.In S. cerevisiae, some 55 members of the binuclear zinc cluster regulator family exist (for a review see Ref. 9). Most exhibit a similar molecular architecture and domain organization. The N-terminal DNA-binding domain is followed by a coiled-coil dimerization domain, by an extended region of limited homology referred to as the "middle homology region" (MHR), 3 and finally by a short acidic stretch encompassing the C-terminal activation domain (10). Fungal zinc cluster pro...