Bettina E. Bauer

Inventory and function of yeast ABC proteins: about sex, stress, pleiotropic drug and heavy metal resistance.

Saccharomyces cerevisiae was the first eukaryotic organism whose complete genome sequence has been determined, uncovering the existence of numerous genes encoding proteins of the ATP-binding cassette (ABC) family. Fungal ABC proteins are implicated in a variety of cellular functions, ranging from clinical drug resistance development, pheromone secretion, mitochondrial function, peroxisome biogenesis, translation elongation, stress response to cellular detoxification. Moreover, some yeast ABC proteins are orthologues of human disease genes, which makes yeast an excellent model system to study the molecular mechanisms of ABC protein-mediated disease. This review provides a comprehensive discussion and update on the function and transcriptional regulation of all known ABC genes from yeasts, including those discovered in fungal pathogens.

War1p, a Novel Transcription Factor Controlling Weak Acid Stress Response in Yeast

ABSTRACT The Saccharomyces cerevisiae ATP-binding cassette (ABC) transporter Pdr12p effluxes weak acids such as sorbate and benzoate, thus mediating stress adaptation. In this study, we identify a novel transcription factor, War1p, as the regulator of this stress adaptation through transcriptional induction of PDR12. Cells lacking War1p are weak acid hypersensitive, since they fail to induce Pdr12p. The nuclear Zn2Cys6 transcriptional regulator War1p forms homodimers and is rapidly phosphorylated upon sorbate stress. The appearance of phosphorylated War1p isoforms coincides with transcriptional activation of PDR12. Promoter deletion analysis identified a novel cis-acting weak acid response element (WARE) in the PDR12 promoter required for PDR12 induction. War1p recognizes and decorates the WARE both in vitro and in vivo, as demonstrated by band shift assays and in vivo footprinting. Importantly, War1p occupies the WARE in the presence and absence of stress, demonstrating constitutive DNA binding in vivo. Our results suggest that weak acid stress triggers phosphorylation and perhaps activation of War1p. In turn, War1p activation is necessary for the induction of PDR12 through a novel signal transduction event that elicits weak organic acid stress adaptation.

The transmembrane domain 10 of the yeast Pdr5p ABC antifungal efflux pump determines both substrate specificity and inhibitor susceptibility.

We have previously shown that a S1360F mutation in transmembrane domain 10 (TMD10) of the Pdr5p ABC transporter modulates substrate specificity and simultaneously leads to a loss of FK506 inhibition. In this study, we have constructed and characterized the S1360F/A/T and T1364F/A/S mutations located in the hydrophilic face of the amphipatic Pdr5p TMD10. A T1364F mutation leads to a reduction in Pdr5p‐mediated azole and rhodamine 6G resistance. Like S1360F, the T1364F and T1364A mutants were nearly non‐responsive to FK506 inhibition. Most remarkably, however, the S1360A mutation increases FK506 inhibitor susceptibility, because Pdr5p–S1360A is hypersensitive to FK506 inhibition when compared with either wild‐type Pdr5p or the non‐responsive S1360F variant. Hence, the Pdr5p TMD10 determines both azole substrate specificity and susceptibility to reversal agents. This is the first demonstration of a eukaryotic ABC transporter where a single residue change causes either a loss or a gain in inhibitor susceptibility, depending on the nature of the mutational change. These results have important implications for the design of efficient reversal agents that could be used to overcome multidrug resistance mediated by ABC transporter overexpression.

Moderately lipophilic carboxylate compounds are the selective inducers of the Saccharomyces cerevisiae Pdr12p ATP‐binding cassette transporter

Saccharomyces cerevisiae displays very strong induction of a single ATP‐binding cassette (ABC) transporter, Pdr12p, when stressed with certain weak organic acids. This is a plasma membrane pump catalysing active efflux of the organic acid anion from the cell. Pdr12p action probably allows S. cerevisiae to maintain lower intracellular levels of several weak organic acid preservatives than would be expected on the basis of the free equilibration of the acid across the cell membrane. This in turn facilitates growth in the presence of these preservatives and therefore yeast spoilage of food materials. Pdr12p appears to confer resistance to those carboxylic acids that, to a reasonable degree, partition into both the lipid bilayer and aqueous phases. Its gene (PDR12) is strongly induced by sorbate, benzoate and certain other moderately lipophilic carboxylate compounds, but not by organic alcohols or high levels of acetate. PDR12 induction reflects the operation of a previously uncharacterized S. cerevisiae stress response, for which the induction signal is probably a high intracellular pool of the organic acid anion. Copyright

Loss of Cmk1 Ca2+–calmodulin-dependent protein kinase in yeast results in constitutive weak organic acid resistance, associated with a post-transcriptional activation of the Pdr12 ATP-binding cassette transporter

Yeast cells display an adaptive stress response when exposed to weak organic acids at low pH. This adaptation is important in the spoilage of preserved foods, as it allows growth in the presence of weak acid food preservatives. In Saccharomyces cerevisiae, this stress response leads to strong induction of the Pdr12 ATP‐binding cassette (ABC) transporter, which catalyses the active efflux of weak acid anions from the cytosol of adapted cells. S. cerevisiae cells lacking the Cmk1 isoform of Ca2+–calmodulin‐dependent protein kinase are intrinsically resistant to weak acid stress, in that they do not need to spend a long adaptive period in lag phase before resuming growth after exposure to this stress. This resistance of the cmk1 mutant is Pdr12 dependent and, unlike with wild‐type S. cerevisiae, cmk1 cells are capable of performing Pdr12‐specific functions such as energy‐dependent cellular extrusion of fluorescein and benzoate. However, they have neither higher PDR12 gene promoter activity nor higher Pdr12 protein levels. The increased Pdr12 activity in cmk1 cells is therefore caused by Cmk1 exerting a negative post‐transcriptional influence over the activity of the Pdr12 ABC transporter, a transporter protein that is constitutively expressed in low‐pH yeast cultures. This is the first preliminary evidence that shows a protein kinase, either directly or indirectly, regulating the activity of a yeast ABC transporter.

Low levels of Ypt protein prenylation cause vesicle polarization defects and thermosensitive growth that can be suppressed by genes involved in cell wall maintenance

The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle‐docking process requires that Ypt proteins are located in the vesicle membrane. C‐terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6‐2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild‐type or mrs6‐2 cells incubated at 25°C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6‐2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.

CHAPTER 14 – INVENTORY AND EVOLUTION OF FUNGAL ABC PROTEIN GENES

All known ABC proteins share a common hallmark domain—the highly conserved ABC domain that is also known as the nucleotide-binding domain (NBD). The NBD contains signature motifs found in all ABC proteins operating from bacteria to man. Membrane-bound ABC proteins also contain variable numbers of membrane-spanning domains arranged in certain membrane architectures. ABC proteins not only function as simple membrane translocators for molecules, they can also act as receptors, sensors, proteases, channels, channel regulators, and even signaling components. This chapter discusses the structure, function, and properties of fungal ABC proteins, focusing on the inventory of ABC genes in S. cerevisiae . Because the functional annotation of the yeast genome is fairly advanced, this chapter also compares the yeast ABC inventory to those from fungal pathogens whose genomes have been sequenced or are close to being sequenced.

Mrs6p, the yeast homologue of the mammalian choroideraemia protein: immunological evidence for its function as the Ypt1p Rab escort protein.

The Saccharomyces cerevisiae MRS6 gene belongs to the same gene family as that responsible for the mammalian Rab escort protein (REP) and the Rab GDP dissociation inhibitor protein (GDI). Both REP and GDI are regulators of the Ras-related small G-proteins Rab/YPT1 which are involved in intracellular vesicular trafficking in yeast and in mammals. Here were characterize an antiserum directed against Mrs6p and show that it specifically inhibits the geranylation of the YPT1 protein in an in vitro assay. These findings provide direct evidence for the role of Mrs6p as the REP component of the yeast Rab geranylgeranyl transferase enzyme.

A genetic screen identifies mutations in the yeast WAR1 gene, linking transcription factor phosphorylation to weak‐acid stress adaptation

Exposure of the yeast Saccharomyces cerevisiae to weak organic acids such as the food preservatives sorbate, benzoate and propionate leads to the pronounced induction of the plasma membrane ATP‐binding cassette (ABC) transporter, Pdr12p. This protein mediates efflux of weak acid anions, which is essential for stress adaptation. Recently, we identified War1p as the dedicated transcriptional regulator required for PDR12 stress induction. Here, we report the results from a genetic screen that led to the isolation of two war1 alleles encoding mutant variants, War1‐28p and War1‐42p, which are unable to support cell growth in the presence of sorbate. DNA sequencing revealed that War1‐28 encodes a truncated form of the transcriptional regulator, and War1‐42 carries three clustered mutations near the C‐terminal activation domain. Although War1‐42 is expressed and properly localized in the nucleus, the War1‐42p variant fails to bind the weak‐acid‐response elements in the PDR12 promoter, as shown by in vivo footprinting. Importantly, in contrast with wild‐type War1p, War1‐42p is also no longer phosphorylated upon weak‐acid challenge, demonstrating that phosphorylation of War1p, its activation and DNA binding are tightly linked processes that are essential for adaptation to weak‐acid stress.

CHAPTER 15 – FUNGAL ABC PROTEINS IN CLINICAL DRUG RESISTANCE AND CELLULAR DETOXIFICATION

Expression of several yeast ABC proteins is linked to, or causes, pleiotropic drug resistance (PDR) phenomena and certain ABC genes represent orthologues of mammalian disease genes. S. cerevisiae is, thus, considered an important model organism to study the function of evolutionary conserved genes, including mammalian ABC proteins of medical importance. The PDR phenomenon is phenotypically quite analogous to multidrug resistance (MDR) as it develops in mammalian cells, parasites, fungal pathogens, or even in bacteria. MDR can be described as an initial resistance to a single drug, followed by cross-resistance to many structurally and functionally unrelated compounds. ABC pumps have been used to identify and clone resistance genes from fungal pathogens such as Candida and Aspergillus species. This chapter is devoted to a comprehensive discussion of ABC protein-mediated drug resistance phenomena as they have been described in model systems like S. cerevisiae as well as in fungal pathogens.