The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.

The vma2⌬ mutant contains elevated levels of reactive oxygen species and high levels of oxidative protein damage even in the absence of an applied oxidant, suggesting an endogenous source of oxidative stress. vma2⌬ mutants lacking mitochondrial DNA showed neither improved growth nor decreased sensitivity to peroxide, excluding respiration as the major source of the endogenous reactive oxygen species in the mutant. Double mutants lacking both VMA2 and components of the major cytosolic defense systems exhibited synthetic sensitivity to H 2 O 2 . Microarray analysis comparing wild-type and vma2⌬ mutant cells grown at pH 5, permissive conditions for the vma2⌬ mutant, indicated high level up-regulation of several iron uptake and metabolism genes that are part of the Aft1/Aft2 regulon. TSA2, which encodes an isoform of the cytosolic thioredoxin peroxidase, was strongly induced, but other oxidative stress defense systems were not induced. The results indicate that V-ATPase activity helps to protect cells from endogenous oxidative stress.Both prokaryotic and eukaryotic cells have multiple mechanisms for preventing and counteracting the effects of oxidative stress (1). Reactive oxygen species (ROS) 3 are normal products of aerobic metabolism, but excess ROS accumulation due to perturbation of control mechanisms can result in accumulation of mutations, premature aging, and cell death (2-4). Many of the central mechanisms for controlling redox balance in the cell are highly conserved. These mechanisms include 1) proteins involved directly in regulating redox balance or repairing effects of oxidative stress, including the well known components of the glutathione-and thioredoxin-dependent reduction systems, superoxide dismutase, catalase, and the pentose phosphate pathway, and 2) a transcriptional response centered around recruitment of the "redox-specific" transcription factors to promoters of these and other antioxidant response genes (2, 3).Only recently has the full spectrum of genes important for control of oxidative stress begun to be appreciated, through microarray analysis of responses to a variety of oxidants and screens of ordered S. cerevisiae deletion mutant arrays (5-7). Genomic screens have revealed that deletions in many genes not previously associated with redox balance result in sensitivity to one or more applied oxidants (6 -8). These screens also revealed that response to oxidative stress may be more specific than previously appreciated, since many deletion mutants show sensitivity to one type of oxidant but little sensitivity to others (7,8). These results suggest that the cell tailors its response to different stresses rather than having a single general response pathway (9). However, certain deletion strains do show sensitivity to multiple different applied oxidants (7,8). This may indicate that the deletions in these genes affect protective mechanisms that are used in multiple contexts and/or that the deletions result in a chronic state of oxidative stress that render the mutants unable to cope with ...

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TAF9 is a TATA-binding protein associated factor (TAF) conserved from yeast to humans and shared by two transcription coactivator complexes, TFIID and SAGA. The essentiality of the TAFs has made it difficult to ascertain their roles in TFIID and SAGA function. Here we performed a genomic synthetic genetic array analysis using a temperature-sensitive allele of TAF9 as a query. Results from this experiment showed that TAF9 interacts genetically with: (1) genes for multiple transcription factor complexes predominantly involving Mediator, chromatin modification/remodeling complexes, and regulators of transcription elongation; (2) virtually all nonessential genes encoding subunits of the SWR-C chromatin-remodeling complex and both TAF9 and SWR-C required for expressing the essential housekeeping gene RPS5; and (3) key genes for cell cycle control at the G 1 /S transition, as well as genes involved in cell polarity, cell integrity, and protein synthesis, suggesting a link between TAF9 function and cell growth control. We also showed that disruption of SAGA by deletion of SPT20 alters histone-DNA contacts and phosphorylated forms of RNA polymerase II at coding sequences. Our results raise the possibility of an unappreciated role for TAF9 in transcription elongation, perhaps in the context of SAGA, and provide further support for TAF9 involvement in cell cycle progression and growth control.

Drug resistance as a result of overexpression of drug transporter genes presents a major obstacle in the treatment of cancers and infections. The molecular mechanisms underlying transcriptional up-regulation of drug transporter genes remains elusive. Employing Saccharomyces cerevisiae as a model, we analyzed here transcriptional regulation of the drug transporter gene PDR5 in a drug-resistant pdr1-3 strain. This mutant bears a gain-of-function mutation in PDR1, which encodes a transcriptional activator for PDR5. Similar to the well studied model gene GAL1, we provide evidence showing that PDR5 belongs to a group of genes whose transcription requires the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex. We also show that the drugindependent PDR5 transcription is associated with enhanced promoter occupancy of coactivator complexes, including SAGA, Mediator, chromatin remodeling SWI/ SNF complex, and TATA-binding protein. Analyzed by chromatin immunoprecipitations, loss of contacts between histones and DNA occurs at both promoter and coding sequences of PDR5. Consistently, micrococcal nuclease susceptibility analysis revealed altered chromatin structure at the promoter and coding sequences of PDR5. Our data provide molecular description of the changes associated with constitutive PDR5 transcription, and reveal the molecular mechanism underlying drug-independent transcriptional up-regulation of PDR5.Transcriptional regulation of the ATP binding cassette (ABC) transporter genes plays a pivotal role in the development of a drug resistance phenotype in mammalian (1, 2) and yeast cells (3-5). Constitutive transcriptional up-regulation of drug transporter genes occurs in certain mutations involving various transcriptional activators. However, the molecular mechanism underlying this enhanced transcription is not fully understood.In Saccharomyces cerevisiae, the multiple/pleiotropic drug resistance (MDR/PDR) phenotype is primarily regulated via two transcriptional activators encoded by homologous PDR1 and PDR3 genes. These factors are responsible for activating the majority of drug transporter genes, including PDR5 (5-7). Both activators belong to the Gal4 superfamily with the Zn2Cys6 DNA binding domain (8). The amino-terminal of their DNA binding domains recognize promoters with the Pdr1/Pdr3 response element (PDRE), 1 5Ј-TCCGCGGA-3Ј (9), such as that with PDR5.The Pdr5 transporter belongs to the ABC transporter superfamily. In the drug-resistant pdr1-3 and pdr3-7 strains, PDR5 transcription is the highest among target genes activated by Pdr1 and Pdr3 (10). The pdr1-3 and pdr3-7 strains are analogous to other mutants bearing gain-of-function mutations in PDR1 (11) and PDR3 (12) that up-regulate PDR5 transcription. The pdr1-3 allele bears a F815S mutation located near the transcription activation domain at the COOH terminus of Pdr1, suggesting that the mutation may alter the function of the activation domain.Activation of transcription in eukaryotes is mainly controlled by activator-mediated recruitments of transcription fact...