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.

Ral is a ubiquitously expressed Ras-like small GTPase which is abundantly present in human platelets. The biological function of Ral and the signaling pathway in which Ral is involved are largely unknown. Here we describe a novel method to measure Ral activation utilizing the Ral binding domain of the putative Ral effector RLIP76 as an activation-specific probe. With this assay we investigated the signaling pathway that leads to Ral activation in human platelets. We found that Ral is rapidly activated after stimulation with various platelet agonists, including ␣-thrombin. In contrast, the platelet antagonist prostaglandin I 2 inhibited ␣-thrombininduced Ral activation. Activation of Ral by ␣-thrombin could be inhibited by depletion of intracellular Ca 2؉ , whereas the induction of intracellular Ca 2؉ resulted in the activation of Ral. Our results show that Ral can be activated by extracellular stimuli. Furthermore, we show that increased levels of intracellular Ca 2؉ are sufficient for Ral activation in platelets. This activation mechanism correlates with the activation mechanism of the small GTPase Rap1, a putative upstream regulator of Ral guanine nucleotide exchange factors.RalA and RalB are very similar small GTPases that have 55% sequence identity with Ras (6,7,41). Ral and Ras have comparable nucleotide binding characteristics and low intrinsic GTPase activity (19). The Ral proteins are ubiquitously expressed but are particularly abundant in brain, testes, and platelets (2, 21, 39). Ral becomes posttranslationally processed and is found in the plasma membrane (16) as well as in endocytotic vesicles (41) or synaptic vesicles (50). In platelets, Ral is a major GTP binding protein that is present in the plasma membrane and specifically in dense granules, a class of secretory organelles (29,41).Recently, a putative effector protein of RalA and RalB has been identified; it has been designated RLIP76 but is also termed RalBP1 or RIP1 (5,22,40). RLIP76 interacts with the active, GTP-bound form of Ral, both in the yeast two-hybrid system and in vitro. Interaction studies using the yeast twohybrid system and deletion mutants of RLIP76 demonstrate that the Ral binding region is located in the C-terminal region, between amino acids 403 and 499 (22). Interestingly, RLIP76 exhibits GTPase-activating protein (GAP) activity for the Rholike GTPase Cdc42, suggesting that Ral may be involved in the regulation of Cdc42 (5, 22, 40). Cdc42 plays a role in the organization of the actin cytoskeleton and the regulation of cytoskeletal polarity.Ral also interacts with phospholipase D1 (PLD1) (21, 28). The interaction between Ral and PLD1 is independent of the nucleotide binding of Ral and occurs via the N-terminal region of Ral (21,28). In platelets and other cells, PLD has been implicated in vesicle transport, regulation of the actin cytoskeleton, and generation of lysophosphatidic acid, which is secreted by platelets upon activation (14, 31).Different proteins that regulate the activity of Ral have been identified. A RalGAP with...

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 Zn 2 Cys 6 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.

Weak organic acids such as sorbate are potent fungistatic agents used in food preservation, but their intracellular targets are poorly understood. We thus searched for potential target genes and signaling components in the yeast genome using contemporary genome-wide functional assays as well as DNA microarray profiling. Phenotypic screening of the EURO-SCARF collection revealed the existence of numerous sorbate-sensitive strains. Sorbate hypersensitivity was detected in mutants of the shikimate biosynthesis pathway, strains lacking the PDR12 efflux pump or WAR1, a transcription factor mediating stress induction of PDR12. Using DNA microarrays, we also analyzed the genome-wide response to acute sorbate stress, allowing for the identification of more than 100 genes rapidly induced by weak acid stress. Moreover, a novel War1p-and Msn2p/4p-independent regulon that includes HSP30 was identified. Although induction of the majority of sorbate-induced genes required Msn2p/4p, weak acid tolerance was unaffected by a lack of Msn2p/4p. Ectopic expression of PDR12 from the GAL1-10 promoter fully restored sorbate resistance in a strain lacking War1p, demonstrating that PDR12 is the major target of War1p under sorbic acid stress. Interestingly, comparison of microarray data with results from the phenotypic screening revealed that PDR12 remained as the only gene, which is both stress inducible and required for weak acid resistance. Our results suggest that combining functional assays with transcriptome profiling allows for the identification of key components in large datasets such as those generated by global microarray analysis.

The ability of yeasts to grow in the presence of weak organic acid preservatives is an important cause of food spoilage. Many of the determinants of acetate resistance in Saccharomyces cerevisiae differ from the determinants of resistance to the more lipophilic sorbate and benzoate. Interestingly, we show in this study that hypersensitivity to both acetate and sorbate results when the cells have auxotrophic requirements for aromatic amino acids. In tryptophan biosynthetic pathway mutants, this weak acid hypersensitivity is suppressed by supplementing the medium with high levels of tryptophan or, in the case of sorbate sensitivity, by overexpressing the Tat2p high affinity tryptophan permease. Weak acid stress therefore inhibits uptake of aromatic amino acids from the medium. This allows auxotrophic requirements for these amino acids to strongly influence the resistance phenotypes of mutant strains. This property must be taken into consideration when using these phenotypes to attribute functional assignments to genes. We show that the acetate sensitivity phenotype previously ascribed to yeast mutants lacking the Pdr12p and Azr1p plasma membrane transporters is an artefact arising from the use of trp1 mutant strains. These transporters do not confer resistance to high acetate levels and, in prototrophs, their presence is actually detrimental for this resistance.