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Macrophage autophagy is a highly anti-atherogenic process that promotes the catabolism of cytosolic lipid droplets (LDs) to maintain cellular lipid homeostasis. Selective autophagy relies on tags such as ubiquitin and a set of selectivity factors including selective autophagy receptors (SARs) to label specific cargo for degradation. Originally described in yeast cells, “lipophagy” refers to the degradation of LDs by autophagy. Yet, how LDs are targeted for autophagy is poorly defined. Here, we employed mass spectrometry to identify lipophagy factors within the macrophage foam cell LD proteome. In addition to structural proteins (e.g., PLIN2), metabolic enzymes (e.g., ACSL) and neutral lipases (e.g., PNPLA2), we found the association of proteins related to the ubiquitination machinery (e.g., AUP1) and autophagy (e.g., HMGB, YWHA/14-3-3 proteins). The functional role of candidate lipophagy factors (a total of 91) was tested using a custom siRNA array combined with high-content cholesterol efflux assays. We observed that knocking down several of these genes, including Hmgb1, Hmgb2, Hspa5, and Scarb2, significantly reduced cholesterol efflux, and SARs SQSTM1/p62, NBR1 and OPTN localized to LDs, suggesting a role for these in lipophagy. Using yeast lipophagy assays, we established a genetic requirement for several candidate lipophagy factors in lipophagy, including HSPA5, UBE2G2 and AUP1. Our study is the first to systematically identify several LD-associated proteins of the lipophagy machinery, a finding with important biological and therapeutic implications. Targeting these to selectively enhance lipophagy to promote cholesterol efflux in foam cells may represent a novel strategy to treat atherosclerosis. Abbreviations: ADGRL3: adhesion G protein-coupled receptor L3; agLDL: aggregated low density lipoprotein; AMPK: AMP-activated protein kinase; APOA1: apolipoprotein A1; ATG: autophagy related; AUP1: AUP1 lipid droplet regulating VLDL assembly factor; BMDM: bone-marrow derived macrophages; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; BSA: bovine serum albumin; CALCOCO2: calcium binding and coiled-coil domain 2; CIRBP: cold inducible RNA binding protein; COLGALT1: collagen beta(1-O)galactosyltransferase 1; CORO1A: coronin 1A; DMA: deletion mutant array; Faa4: long chain fatty acyl-CoA synthetase; FBS: fetal bovine serum; FUS: fused in sarcoma; HMGB1: high mobility group box 1; HMGB2: high mobility group box 2: HSP90AA1: heat shock protein 90: alpha (cytosolic): class A member 1; HSPA5: heat shock protein family A (Hsp70) member 5; HSPA8: heat shock protein 8; HSPB1: heat shock protein 1; HSPH1: heat shock 105kDa/110kDa protein 1; LDAH: lipid droplet associated hydrolase; LIPA: lysosomal acid lipase A; LIR: LC3-interacting region; MACROH2A1: macroH2A.1 histone; MAP1LC3: microtubule-associated protein 1 light chain 3; MCOLN1: mucolipin 1; NBR1: NBR1, autophagy cargo receptor; NPC2: NPC intracellular cholesterol transporter 2; OPTN: optineurin; P/S: penicillin-streptomycin; PLIN2: perilipin 2; PLIN3: perilipin 3; PNPLA2: patatin like phospholipase domain containing 2; RAB: RAB, member RAS oncogene family; RBBP7, retinoblastoma binding protein 7, chromatin remodeling factor; SAR: selective autophagy receptor; SCARB2: scavenger receptor class B, member 2; SGA: synthetic genetic array; SQSTM1: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; TFEB: transcription factor EB; TOLLIP: toll interacting protein; UBE2G2: ubiquitin conjugating enzyme E2 G2; UVRAG: UV radiation resistance associated gene; VDAC2: voltage dependent anion channel 2; VIM: vimentin

Acetyl-CoA Carboxylase 1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step of de novo fatty acid synthesis. As a master regulator of lipid synthesis, acetyl-CoA carboxylase 1 has been proposed to be a therapeutic target for numerous metabolic diseases. We have shown that acetyl-CoA carboxylase 1 activity is reduced in the absence of the lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This change in acetyl-CoA carboxylase 1 activity is correlated with a change in localization. In wild-type cells, acetyl-CoA carboxylase 1 is localized throughout the cytoplasm in small punctate and rod-like structures. However, in NuA4 mutants, acetyl-CoA carboxylase 1 localization becomes diffuse. To uncover mechanisms regulating acetyl-CoA carboxylase 1 localization, we performed a microscopy screen to identify other deletion mutants that impact acetyl-CoA carboxylase 1 localization and then measured acetyl-CoA carboxylase 1 activity in these mutants through chemical genetics and biochemical assays. Three phenotypes were identified. Mutants with hyper-active acetyl-CoA carboxylase 1 form 1 or 2 rod-like structures centrally within the cytoplasm, mutants with mid-low acetyl-CoA carboxylase 1 activity displayed diffuse acetyl-CoA carboxylase 1, while the mutants with the lowest acetyl-CoA carboxylase 1 activity (hypomorphs) formed thick rod-like acetyl-CoA carboxylase 1 structures at the periphery of the cell. All the acetyl-CoA carboxylase 1 hypomorphic mutants were implicated in sphingolipid metabolism or very long-chain fatty acid elongation and in common, their deletion causes an accumulation of palmitoyl-CoA. Through exogenous lipid treatments, enzyme inhibitors, and genetics, we determined that increasing palmitoyl-CoA levels inhibits acetyl-CoA carboxylase 1 activity and remodels acetyl-CoA carboxylase 1 localization. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of acetyl-CoA carboxylase 1 can fine-tune the rate of fatty acid synthesis.

Under environmental stress, such as glucose deprivation, cells form stress granules - the accumulation of cytoplasmic aggregates of repressed translational initiation complexes, proteins, and stalled mRNAs. Recent research implicates stress granules in various diseases, such as neurodegenerative diseases, but the exact regulators responsible for the assembly and disassembly of stress granules are unknown. An important aspect of stress granule formation is the presence of post-translational modifications on core proteins. One of those modifications is lysine acetylation, which is regulated by either a lysine acetyltransferase (KAT) or a lysine deacetylase (KDAC) enzyme. This work deciphers the impact of lysine acetylation on an essential protein found in Saccharomyces cerevisiae stress granules, poly(A) binding protein (Pab1). We demonstrated that an acetylation mimic of the lysine residue in the position 131 reduces stress granule formation upon glucose deprivation and other stressors such as ethanol, raffinose, and vanillin. We present genetic evidence that the enzyme Rpd3 is the primary candidate for deacetylation of Pab1-K131. Further, our electromobility shift assay studies suggest that the acetylation of Pab1-K131 negatively impacts poly(A) RNA binding. Due to the conserved nature of stress granules, therapeutics targeting the activity of KATs and KDAC enzymes may be a promising route to modulate stress granule dynamics in the disease state.

Summary Adipic acid production by yeast fermentation is gaining attention as a renewable source of platform chemicals for making nylon products. However, adipic acid toxicity inhibits yeast growth and fermentation. Here, we performed a chemogenomic screen in Saccharomyces cerevisiae to understand the cellular basis of adipic acid toxicity. Our screen revealed that KGD1 (a key gene in the tricarboxylic acid cycle) deletion improved tolerance to adipic acid and its toxic precursor, catechol. Conversely, disrupting ergosterol biosynthesis as well as protein trafficking and vacuolar transport resulted in adipic acid hypersensitivity. Notably, we show that adipic acid disrupts the Membrane Compartment of Can1 (MCC) on the plasma membrane and impacts endocytosis. This was evidenced by the rapid internalization of Can1 for vacuolar degradation. As ergosterol is an essential component of the MCC and protein trafficking mechanisms are required for endocytosis, we highlight the importance of these cellular processes in modulating adipic acid toxicity., Graphical abstract, Highlights • Deletion of the TCA cycle gene KGD1 improves tolerance to adipic acid and catechol • Ergosterol and Pdr12 play non-overlapping roles protecting cell from adipic acid • Adipic acid-induced plasma membrane localization of Pdr12 is independent of ergosterol • Adipic acid disrupts the Membrane Compartment of Can1 (MCC) and induces endocytosis, Cell Biology; Systems Biology

Cellular metabolism is tightly regulated by many signaling pathways and processes, including lysine acetylation of proteins. While lysine acetylation of metabolic enzymes can directly influence enzyme activity, there is growing evidence that lysine acetylation can also impact protein localization. As the Saccharomyces cerevisiae lysine acetyltransferase complex NuA4 has been implicated in a variety of metabolic processes, we have explored whether NuA4 controls the localization and/or protein levels of metabolic proteins. We performed a high-throughput microscopy screen of over 360 GFP-tagged metabolic proteins and identified 23 proteins whose localization and/or abundance changed upon deletion of the NuA4 scaffolding subunit, EAF1. Within this, three proteins were required for glycogen synthesis and 14 proteins were associated with the mitochondria. We determined that in eaf1Δ cells the transcription of glycogen biosynthesis genes is upregulated resulting in increased proteins and glycogen production. Further, in the absence of EAF1, mitochondria are highly fused, increasing in volume approximately 3-fold, and are chaotically distributed but remain functional. Both the increased glycogen synthesis and mitochondrial elongation in eaf1Δ cells are dependent on Bcy1, the yeast regulatory subunit of PKA. Surprisingly, in the absence of EAF1, Bcy1 localization changes from being nuclear to cytoplasmic and PKA activity is altered. We found that NuA4-dependent localization of Bcy1 is dependent on a lysine residue at position 313 of Bcy1. However, the glycogen accumulation and mitochondrial elongation phenotypes of eaf1Δ, while dependent on Bcy1, were not fully dependent on Bcy1-K313 acetylation state and subcellular localization of Bcy1. As NuA4 is highly conserved with the human Tip60 complex, our work may inform human disease biology, revealing new avenues to investigate the role of Tip60 in metabolic diseases., Author summary Metabolism, how cells process nutrients and energy substrates, is dramatically altered in many diseases. For example, during tumorigenesis, changes in cellular metabolism allow for the rapid cellular growth and division. One way to control metabolism is by moving the proteins involved in metabolism to different parts of the cell. Similar to an assembly line, when all the “parts” (metabolic proteins) are brought together, efficiency can be increased. In contrast, if one or more parts are shipped to the wrong place (mislocalized), production is decreased, and an alternative production process may even be necessary to meet demands. Here we ask whether Tip60, a protein complex implicated in many diseases, affects the location of metabolic proteins in the cell. We use budding yeast as a model system to assess the effect of disrupting yeast Tip60 on the location of over 360 metabolic proteins. We found that Tip60 controls the location of many metabolic proteins and in the absence of Tip60 there is an increase in the mitochondria and in cellular carbohydrate storage (the metabolic ‘hubs’ of the cell). This work suggests that yeast Tip60 functions as a “brake” on cellular metabolism and provides novel potential roles of Tip60 in metabolic diseases.

Synthetic biology has played a major role in engineering microbial cell factories to convert plant biomass (lignocellulose) to fuels and bioproducts by fermentation. However, the final product yield is limited by inhibition of microbial growth and fermentation by toxic phenolic compounds generated during lignocellulosic pre-treatment and hydrolysis. Advances in the development of systems biology technologies (genomics, transcriptomics, proteomics, metabolomics) have rapidly resulted in large datasets which are necessary to obtain a holistic understanding of complex biological processes underlying phenolic compound toxicity. Here, we review and compare different systems biology tools that have been utilized to identify molecular mechanisms that modulate phenolic compound toxicity in Saccharomyces cerevisiae. By focusing on and comparing functional genomics and transcriptomics approaches we identify common mechanisms potentially underlying phenolic toxicity. Additionally, we discuss possible ways by which integration of data obtained across multiple unbiased approaches can result in new avenues to develop yeast strains with a significant improvement in tolerance to phenolic fermentation inhibitors.

The conversion of plant material into biofuels and high value products is a two-step process of hydrolysing plant lignocellulose and next fermenting the sugars produced. However, lignocellulosic hydrolysis not only frees sugars for fermentation it simultaneously generates toxic chemicals, including phenolic compounds which severely inhibit yeast fermentation. To understand the molecular basis of phenolic compound toxicity, we performed genome-wide chemogenomic screens in Saccharomyces cerevisiae to identify deletion mutants that were either hypersensitive or resistant to three common phenolic compounds found in plant hydrolysates: coniferyl aldehyde, ferulic acid and 4-hydroxybenzoic acid. Despite being similar in structure, our screen revealed that yeast utilizes distinct pathways to tolerate phenolic compound exposure. Furthermore, although each phenolic compound induced reactive oxygen species (ROS), ferulic acid and 4-hydroxybenzoic acid-induced a general cytoplasmic ROS distribution while coniferyl aldehyde-induced ROS partially localized to the mitochondria and to a lesser extent, the endoplasmic reticulum. We found that the glucose-6-phosphate dehydrogenase enzyme Zwf1, which catalyzes the rate limiting step of pentose phosphate pathway, is required for reducing the accummulation of coniferyl aldehyde-induced ROS, potentially through the sequestering of Zwf1 to sites of ROS accumulation. Our novel insights into biological impact of three common phenolic inhibitors will inform the engineering of yeast strains with improved efficiency of biofuel and biochemical production in the presence hydrolysate-derived phenolic compounds.

Kes1/Osh4 is a member of the conserved, but functionally enigmatic, oxysterol binding protein-related protein (ORP) superfamily that inhibits phosphatidylinositol transfer protein (Sec14)-dependent membrane trafficking through the trans-Golgi (TGN)/endosomal network. We now report that Kes1, and select other ORPs, execute cell-cycle control activities as functionally non-redundant inhibitors of the G(1)/S transition when cells confront nutrient-poor environments and promote replicative aging. Kes1-dependent cell-cycle regulation requires the Great-wall/MASTL kinase ortholog Rim15, and is opposed by Sec14 activity in a mechanism independent of Kes1/Sec14 bulk membrane-trafficking functions. Moreover, the data identify Kes1 as a non-histone target for NuA4 through which this lysine acetyltransferase co-modulates membrane-trafficking and cell-cycle activities. We propose the Sec14/Kes1 lipid-exchange protein pair constitutes part of the mechanism for integrating TGN/endosomal lipid signaling with cell-cycle progression and hypothesize that ORPs define a family of stage-specific cell-cycle control factors that execute tumor-suppressor-like functions.

Acetylation is a dynamic post-translational modification that is attached to protein substrates by lysine acetyltransferases (KATs) and removed by lysine deacetylases (KDACs). While these enzymes are best characterized as histone modifiers and regulators of gene transcription, work in a number of systems highlights that acetylation is a pervasive modification and suggests a broad scope for KAT and KDAC functions in the cell. As we move beyond generating lists of acetylated proteins, the acetylation field is in dire need of robust tools to connect acetylation and deacetylation machineries to their respective substrates and to dissect the function of individual sites. The Saccharomyces cerevisiae model system provides such a toolkit in the context of both tried and true genetic techniques and cutting-edge proteomic and cell imaging methods. Here, we review these methods in the context of their contributions to acetylation research thus far and suggest strategies for addressing lingering questions in the field.

Many asymmetrically dividing cells unequally partition cellular structures according to age. Yet, it is unclear how cells differentiate pre-existing from newly synthesized material. Yeast cells segregate the spindle pole body (SPB, centrosome equivalent) inherited from the previous mitosis to the bud, while keeping the new one in the mother cell. Here, we show that the SPB inheritance network (SPIN), comprising the kinases Swe1 (also known as Wee1) and Kin3 (also known as Nek2) and the acetyltransferase NuA4 (also known as Tip60), distinguishes pre-existing from new SPBs. Swe1 phosphorylated Nud1 (orthologous to Centriolin) on young SPBs as they turned into pre-existing ones. The subsequent inactivation of Swe1 protected newly assembling SPBs from being marked. Kin3 and NuA4 maintained age marks on SPBs through following divisions. Downstream of SPIN, the Hippo regulator Bfa1-Bub2 bound the marked SPB, directed the spindle-positioning protein Kar9 towards it and drove its partition to the bud. Thus, coordination of SPIN activity and SPB assembly encodes age onto SPBs to enable their age-dependent segregation.

Eukaryotic cells form stress granules under a variety of stresses, however the signaling pathways regulating their formation remain largely unknown. We have determined that the Saccharomyces cerevisiae lysine acetyltransferase complex NuA4 is required for stress granule formation upon glucose deprivation but not heat stress. Further, the Tip60 complex, the human homolog of the NuA4 complex, is required for stress granule formation in cancer cell lines. Surprisingly, the impact of NuA4 on glucose-deprived stress granule formation is partially mediated through regulation of acetyl-CoA levels, which are elevated in NuA4 mutants. While elevated acetyl-CoA levels suppress the formation of glucose-deprived stress granules, decreased acetyl-CoA levels enhance stress granule formation upon glucose deprivation. Further our work suggests that NuA4 regulates acetyl-CoA levels through the Acetyl-CoA carboxylase Acc1. Altogether this work establishes both NuA4 and the metabolite acetyl-CoA as critical signaling pathways regulating the formation of glucose-deprived stress granules., Author summary In response to environmental stress, such as nutrient limitations or toxic chemicals, cells must quickly counteract these threats in order to survive. One way cells fight environmental challenges is through the formation of stress granules, which are aggregates of proteins and mRNA within the cytoplasm. Though their formation is essential for survival under multiple conditions, when stress granules are inappropriately formed they become causative for diseases such as amyotrophic lateral sclerosis and fragile X syndrome. Further stress granules contribute to chemotherapy resistance of cancer cells by promoting survival. Therefore it is critical to understand how stress granules are formed and disassembled. Here using the budding yeast Saccharomyces cerevisiae we determine that an enzyme called NuA4 is contributing to stress granule formation upon glucose deprivation. Why is this important? We determine that Tip60, the equivalent of NuA4 in mammalian cells, is also regulating stress granule formation in cancer cells. As there is a growing number of drugs that target this class of enzyme, there is the possibility that these drugs may reduce stress granule formation offering a novel therapeutic approach to treat numerous diseases.

Fundamental changes in the composition and distribution of lipids within the brain are believed to contribute to the cognitive decline associated with Alzheimer’s disease (AD). The mechanisms by which these changes in lipid composition affect cellular function and ultimately cognition are not well understood. Although “candidate gene” approaches can provide insight into the effects of dysregulated lipid metabolism they require a preexisting understanding of the molecular targets of individual lipid species. In this report we combine unbiased gene expression profiling with a genome-wide chemogenomic screen to identify the mitochondria as an important downstream target of PC(O-16:0/2:0), a neurotoxic lipid species elevated in AD. Further examination revealed that PC(O-16:0/2:0) similarly promotes a global increase in ceramide accumulation in human neurons which was associated with mitochondrial-derived reactive oxygen species (ROS) and toxicity. These findings suggest that PC(O-16:0/2:0)-dependent mitochondrial dysfunction may be an underlying contributing factor to the ROS production associated with AD.

The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1 in other cellular processes independent of iron regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1 interacts with and co-localizes with kinetochore proteins; however, the cellular implications of this have not been established. Here, we demonstrate that Aft1 associates with the kinetochore complex through Iml3. Furthermore, like Iml3, Aft1 is required for the increased association of cohesin with pericentric chromatin, which is required to resist microtubule tension, and aft1Δ cells display chromosome segregation defects in meiosis. Our work defines a new role for Aft1 in chromosome stability and transmission.

To paraphrase Robert Burns's poem To a Mouse, the best laid schemes of DNA-protein complex purification often go awry. Chromatin with its heterogeneous and dynamic protein composition remains difficult to analyze. Still critical progress has been made in recent years in characterizing the interface between DNA and proteins due, in part, to significant advances in proteomic technologies. Proteomics has progressed to a point where affinity purification of soluble complexes and protein identification by mass spectrometry are routine. The new challenge for chromatin proteomics lies in studying proteins and protein complexes in their native environment, which is on chromatin. These novel types of data represent an additional layer of information that can be used to better characterize and understand cellular processes. This review will focus on the past contributions as well as on emerging mass spectrometry-based methodologies attempting to better define the complex relationship between proteins, protein complexes and DNA.

Protein-protein interaction mapping has progressed rapidly in recent years, enabling the completion of several high throughput studies. However, knowledge of physical interactions is limited for numerous classes of proteins, such as chromatin-bound proteins, because of their poor solubility when bound to DNA. To address this problem, we have developed a novel method, termed modified chromatin immunopurification (mChIP), that allows for the efficient purification of protein-DNA macromolecules, enabling subsequent protein identification by mass spectrometry. mChIP consists of a single affinity purification step whereby chromatin-bound protein networks are isolated from mildly sonicated and gently clarified cellular extracts using magnetic beads coated with antibodies. We applied the mChIP method in Saccharomyces cerevisiae cells expressing endogenously tandem affinity purification (TAP)-tagged histone H2A or the histone variant Htz1p and successfully co-purified numerous chromatin-bound protein networks as well as DNA. We further challenged the mChIP procedure by purifying three chromatin-bound bait proteins that have proven difficult to purify by traditional methods: Lge1p, Mcm5p, and Yta7p. The protein interaction networks of these three baits dramatically expanded our knowledge of their chromatin environments and illustrate that the innovative mChIP procedure enables an improved characterization of chromatin-associated proteins.

The SAS3-dependent NuA3 histone acetyltransferase complex was originally identified on the basis of its ability to acetylate histone H3 in vitro. Whether NuA3 is capable of acetylating histones in vivo, or how the complex is targeted to the nucleosomes that it modifies, was unknown. To address this question, we asked whether NuA3 is associated with chromatin in vivo and how this association is regulated. With a chromatin pulldown assay, we found that NuA3 interacts with the histone H3 amino-terminal tail, and loss of the H3 tail recapitulates phenotypes associated with loss of SAS3. Moreover, mutation of histone H3 lysine 14, the preferred site of acetylation by NuA3 in vitro, phenocopies a unique sas3Delta phenotype, suggesting that modification of this residue is important for NuA3 function. The interaction of NuA3 with chromatin is dependent on the Set1p and Set2p histone methyltransferases, as well as their substrates, histone H3 lysines 4 and 36, respectively. These results confirm that NuA3 is functioning as a histone acetyltransferase in vivo and that histone H3 methylation provides a mark for the recruitment of NuA3 to nucleosomes.

The vast accumulation of knowledge from genome sequencing projects and studies with model organisms has presented a remarkable challenge to biologists: to understand the functions of thousands of highly conserved genes and how they work together to regulate fundamental cellular processes. This challenge is compounded by the inescapable reality that most genes are 'buffered' by other genes that contribute to the same biological processes, limiting the impact of phenotypic studies with single mutants. In budding yeast, functional genomic methods have been developed for the systematic application of established genetic techniques. In particular, the Synthetic Genetic Array (SGA) method allows genome-wide synthetic lethal (SL) and synthetic dosage lethal (SDL) screens thus enabling an unbiased survey of genetic interactions. We have used genes encoding components of the yeast kinetochore as a biological testbed for assaying the utility of SGA-based SL and SDL screens for revealing new pathways and genes involved in chromosome segregation. We identified 211 nonessential deletion mutants that were unable to tolerate either overexpression or loss of function of kinetochore genes. Our study uncovered a wealth of relationships between gene products that functionally interact with the kinetochore, and also highlighted the value of performing genome-wide screens with both hypomorphic and hypermorphic alleles of query genes. Here, we will highlight our recent kinetochore SGA genomic screens, in the broader context of applying complementary genetic screening approaches in the systematic exploration of biological pathways or functional complexes.

NuA4, the only essential histone acetyltransferase complex in Saccharomyces cerevisiae , acetylates the N-terminal tails of histones H4 and H2A. Affinity purification of NuA4 revealed the presence of three previously undescribed subunits, Vid21/Eaf1/Ydr359c, Swc4/Eaf2/Ygr002c, and Eaf7/Ynl136w. Experimental analyses revealed at least two functionally distinct sets of polypeptides in NuA4: ( i ) Vid21 and Yng2, and ( ii ) Eaf5 and Eaf7. Vid21 and Yng2 are required for bulk histone H4 acetylation and are functionally linked to the histone H2A variant Htz1 and the Swr1 ATPase complex (SWR-C) that assembles Htz1 into chromatin, whereas Eaf5 and Eaf7 have a different, as yet undefined, role. Mutations in Htz1, the SWR-C, and NuA4 cause defects in chromosome segregation that are consistent with genetic interactions we have observed between the genes encoding these proteins and genes encoding kinetochore components. Because SWR-C-dependent recruitment of Htz1 occurs in both transcribed and centromeric regions, a NuA4/SWR-C/Htz1 pathway may regulate both transcription and centromere function in S. cerevisiae .

Gene expression noise varies with genomic position and is a driving force in the evolution of chromosome organization. Nevertheless, position effects remain poorly characterized. Here, we present a systematic analysis of chromosomal position effects by characterizing single-cell gene expression from euchromatic positions spanning the length of a eukaryotic chromosome. We demonstrate that position affects gene expression by modulating the size of transcriptional bursts, rather than their frequency, and that the histone deacetylase Sir2 plays a role in this process across the chromosome.

Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-β42 signals the intraneuronal accumulation of PC(O-16:0/2:0) which is associated with neurotoxicity. Similar to neuronal cells, intracellular accumulation of PC(O-16:0/2:0) is also toxic to Saccharomyces cerevisiae, making yeast an excellent model to decipher the pathological effects of this lipid. We previously reported that phospholipase D, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)-binding protein, was relocalized in response to PC(O-16:0/2:0), suggesting that this neurotoxic lipid may remodel lipid signaling networks. Here we show that PC(O-16:0/2:0) regulates the distribution of the PtdIns(4)P 5-kinase Mss4 and its product PtdIns(4,5)P2 leading to the formation of invaginations at the plasma membrane (PM). We further demonstrate that the effects of PC(O-16:0/2:0) on the distribution of PM PtdIns(4,5)P2 pools are in part mediated by changes in the biosynthesis of long chain bases (LCBs) and ceramides. A combination of genetic, biochemical and cell imaging approaches revealed that PC(O-16:0/2:0) is also a potent inhibitor of signaling through the Target of rampamycin complex 2 (TORC2). Together, these data provide mechanistic insight into how specific disruptions in phosphocholine second messenger metabolism associated with Alzheimer's disease may trigger larger network-wide disruptions in ceramide and phosphoinositide second messenger biosynthesis and signaling which have been previously implicated in disease progression., Author Summary Accelerated cognitive decline in Alzheimer's patients is associated with distinct changes in the abundance of choline-containing lipids belonging to the platelet activating factor family. In particular, PC(O-16:0/2:0) or C16:0 platelet activating factor (PAF), is specifically elevated in brains of Alzheimer's patients. Since elevated intraneuronal levels of PC(O-16:0/2:0) are thought to contribute to the loss of neuronal cells it is imperative to identify the underlying mechanisms contributing to the toxic effects of PC(O-16:0/2:0). In this study, we have determined that elevated levels of PC(O-16:0/2:0) has negative effects upon the distribution of phosphoinositides at the plasma membrane leading to a potent inhibition of target of rapamycin (TOR) signaling. We further show that the changes in phosphoinositide distribution are due to changes in ceramide metabolism. In conclusion, our study suggests that the toxicity associated with aberrant metabolism of glycerophosphocholine lipids species is likely due to the remodeling of phosphoinositide and ceramide metabolism and that therapeutic strategies which target these disruptions may be effective in ameliorating Alzheimer's Disease pathology.

In Saccharomyces cerevisiae, the heterodimeric transcription factor SBF (for SCB binding factor) is composed of Swi4 and Swi6 and activates gene expression at the G(1)/S-phase transition of the mitotic cell cycle. Cell cycle commitment is associated not only with major alterations in gene expression but also with highly polarized cell growth; the mitogen-activated protein kinase (MAPK) Slt2 is required to maintain cell wall integrity during periods of polarized growth and cell wall stress. We describe experiments aimed at defining the regulatory pathway involving the cell cycle transcription factor SBF and Slt2-MAPK. Gene expression assays and chromatin immunoprecipitation experiments revealed Slt2-dependent recruitment of SBF to the promoters of the G(1) cyclins PCL1 and PCL2 after activation of the Slt2-MAPK pathway. We performed DNA microarray analysis and identified other genes whose expression was reduced in both SLT2 and SWI4 deletion strains. Genes that are sensitive to both Slt2 and Swi4 appear to be uniquely regulated and reveal a role for Swi4, the DNA-binding component of SBF, which is independent of the regulatory subunit Swi6. Some of the Swi4- and Slt2-dependent genes do not require Swi6 for either their expression or for Swi4 localization to their promoters. Consistent with these results, we found a direct interaction between Swi4 and Slt2. Our results establish a new Slt2-dependent mode of Swi4 regulation and suggest roles for Swi4 beyond its prominent role in controlling cell cycle transcription.

In Saccharomyces cerevisiae, two transcription factors, SBF (SCB binding factor) and MBF (MCB binding factor), promote the induction of gene expression at the G(1)/S-phase transition of the mitotic cell cycle. Swi4 and Mbp1 are the DNA binding components of SBF and MBF, respectively. The Swi6 protein is a common subunit of both transcription factors and is presumed to play a regulatory role. SBF binding to its target sequences, the SCBs, is a highly regulated event and requires the association of Swi4 with Swi6 through their C-terminal domains. Swi4 binding to SCBs is restricted to the late M and G(1) phases, when Swi6 is localized to the nucleus. We show that in contrast to Swi6, Swi4 remains nuclear throughout the cell cycle. This finding suggests that the DNA binding domain of Swi4 is inaccessible in the full-length protein when not complexed with Swi6. To explore this hypothesis, we expressed Swi4 and Swi6 in insect cells by using the baculovirus system. We determined that partially purified Swi4 cannot bind SCBs in the absence of Swi6. However, Swi4 derivatives carrying point mutations or alterations in the extreme C terminus were able to bind DNA or activate transcription in the absence of Swi6, and the C terminus of Swi4 inhibited Swi4 derivatives from binding DNA in trans. Full-length Swi4 was determined to be monomeric in solution, suggesting an intramolecular mechanism for auto-inhibition of binding to DNA by Swi4. We detected a direct in vitro interaction between a C-terminal fragment of Swi4 and the N-terminal 197 amino acids of Swi4, which contain the DNA binding domain. Together, our data suggest that intramolecular interactions involving the C-terminal region of Swi4 physically prevent the DNA binding domain from binding SCBs. The interaction of the carboxy-terminal region of Swi4 with Swi6 alleviates this inhibition, allowing Swi4 to bind DNA.

Protein kinase C (PKC) signaling is highly conserved among eukaryotes and has been implicated in the regulation of cellular processes such as cell proliferation and growth. In the budding yeast, PKC1 functions to activate the SLT2(MPK1) mitogen-activated protein (MAP) kinase cascade, which is required for the maintenance of cell integrity during asymmetric cell growth. Genetic studies, coimmunoprecipitation experiments, and analysis of protein phosphorylation in vivo and in vitro indicate that the SBF transcription factor (composed of Swi4p and Swi6p), an important regulator of gene expression at the G 1 to S phase cell cycle transition, is a target of the Slt2p(Mpk1p) MAP kinase. These studies provide evidence for a direct role of the PKC1 pathway in the regulation of the yeast cell cycle and cell growth and indicate that conserved signaling pathways can act to control key regulators of cell division.

Recent global proteomic and genomic studies have determined that lysine acetylation is a highly abundant posttranslational modification. The next challenge is connecting lysine acetyltransferases (KATs) to their cellular targets. We hypothesize that proteins that physically interact with KATs may not only predict the cellular function of the KATs but may be acetylation targets. We have developed a mass spectrometry-based method that generates a KAT protein interaction network from which we simultaneously identify both in vivo acetylation sites and in vitro acetylation sites. This modified chromatin-immunopurification coupled to an in vitro KAT assay with mass spectrometry (mChIP-KAT-MS) was applied to the Saccharomyces cerevisiae KAT nucleosome acetyltransferase of histone H4 (NuA4). Using mChIP-KAT-MS, we define the NuA4 interactome and in vitro-enriched acetylome, identifying over 70 previously undescribed physical interaction partners for the complex and over 150 acetyl lysine residues, of which 108 are NuA4-specific in vitro sites. Through this method we determine NuA4 acetylation of its own subunit Epl1 is a means of self-regulation and identify a unique link between NuA4 and the spindle pole body. Our work demonstrates that this methodology may serve as a valuable tool in connecting KATs with their cellular targets.

CD8+ cytotoxic T lymphocyte (CTL) clones begin to synthesize the lytic proteins granzyme A, granzyme B and perforin after stimulation with allogeneic target cells. The lytic proteins are stored in the secretory granules which are released after cross-linking of the T cell receptor (TcR) upon target cell recognition. During lytic granule biogenesis granzyme A protein synthesis can be detected between 2 and 10 days after allogeneic stimulation of the CTL. Although granzyme A is stored in the lytic granules over this period, the majority of granzyme A synthesized is secreted directly from the CTL. TcR triggering of degranulation also results in new synthesis of the lytic proteins, which can be inhibited by cycloheximide (CHX). Some of the newly synthesized lytic proteins can be stored in the cell and refill the granules. But up to one third of granzymes A and B can be secreted directly from the CTL via the constitutive secretory pathway as shown by granzyme A enzymatic activity and immunoblots of secreted granzyme B, where one third of the protein fails to acquire the granule targeting signal. Perforin is also secreted via the constitutive pathway, both from the natural killer cell line, YT, and from CTL clones after TcR cross-linking. Constitutive secretion of the lytic proteins can be blocked by both CHX and brefeldin A (BFA). While BFA does not affect the directional killing of recognized targets, it abrogates bystander killing, indicating that bystander killing arises from newly synthesized lytic proteins delivered via a non-granule route. These results demonstrate that the perforin/granzyme-mediated lytic pathway can be maintained while CTL kill multiple targets. We show that CTL not only re-fill their granules during killing, but also secrete lytic proteins via a non-granule-mediated pathway.

The variable domains of immunoglobulins and T cell receptors are assembled through the somatic, site specific recombination of multiple germline segments (V, D, and J segments) or V(D)J rearrangement. The recombination signal sequence (RSS) is necessary and sufficient for cell type specific targeting of the V(D)J rearrangement machinery to these germline segments. Previously, the RSS has been described as possessing both a conserved heptamer and a conserved nonamer motif. The heptamer and nonamer motifs are separated by a 'spacer' that was not thought to possess significant sequence conservation, however the length of the spacer could be either 12 +/- 1 bp or 23 +/- 1 bp long. In this report we have assembled and analyzed an extensive data base of published RSS. We have derived, through extensive consensus comparison, a more detailed description of the RSS than has previously been reported. Our analysis indicates that RSS spacers possess significant conservation of sequence, and that the conserved sequence in 12 bp spacers is similar to the conserved sequence in the first half of 23 bp spacers.

Though highly efficient at fermenting hexose sugars, Saccharomyces cerevisiae has limited ability to ferment five-carbon sugars. As a significant portion of sugars found in cellulosic biomass is the five-carbon sugar xylose, S. cerevisiae must be engineered to metabolize pentose sugars, commonly by the addition of exogenous genes from xylose fermenting fungi. However, these recombinant strains grow poorly on xylose and require further improvement through rational engineering or evolutionary adaptation. To identify unknown genes that contribute to improved xylose fermentation in these recombinant S. cerevisiae, we performed genome-wide synthetic interaction screens to identify deletion mutants that impact xylose utilization of strains expressing the xylose isomerase gene XYLA from Piromyces sp. E2 alone or with an additional copy of the endogenous xylulokinase gene XKS1. We also screened the deletion mutant array to identify mutants whose growth is affected by xylose. Our genetic network reveals that more than 80 nonessential genes from a diverse range of cellular processes impact xylose utilization. Surprisingly, we identified four genes, ALP1, ISC1, RPL20B, and BUD21, that when individually deleted improved xylose utilization of both S. cerevisiae S288C and CEN.PK strains. We further characterized BUD21 deletion mutant cells in batch fermentations and found that they produce ethanol even the absence of exogenous XYLA. We have demonstrated that the ability of laboratory strains of S. cerevisiae to utilize xylose as a sole carbon source is suppressed, which implies that S. cerevisiae may not require the addition of exogenous genes for efficient xylose fermentation.

In the budding yeast Saccharomyces cerevisiae, the lysine acetyltransferase NuA4 has been linked to a host of cellular processes through the acetylation of histone and non-histone targets. To discover proteins regulated by NuA4-dependent acetylation, we performed genome-wide synthetic dosage lethal screens to identify genes whose overexpression is toxic to non-essential NuA4 deletion mutants. The resulting genetic network identified a novel link between NuA4 and septin proteins, a group of highly conserved GTP-binding proteins that function in cytokinesis. We show that acetyltransferase-deficient NuA4 mutants have defects in septin collar formation resulting in the development of elongated buds through the Swe1-dependent morphogenesis checkpoint. We have discovered multiple sites of acetylation on four of the five yeast mitotic septins, Cdc3, Cdc10, Cdc12 and Shs1, and determined that NuA4 can acetylate three of the four in vitro. In vivo we find that acetylation levels of both Shs1 and Cdc10 are reduced in a catalytically inactive esa1 mutant. Finally, we determine that cells expressing a Shs1 protein with decreased acetylation in vivo have defects in septin localization that are similar to those observed in NuA4 mutants. These findings provide the first evidence that yeast septin proteins are acetylated and that NuA4 impacts septin dynamics.

The Saccharomyces cerevisiae transcription factor Aft1 is activated in iron-deficient cells to induce the expression of iron regulon genes, which coordinate the increase of iron uptake and remodel cellular metabolism to survive low-iron conditions. In addition, Aft1 has been implicated in numerous cellular processes including cell-cycle progression and chromosome stability; however, it is unclear if all cellular effects of Aft1 are mediated through iron homeostasis. To further investigate the cellular processes affected by Aft1, we identified >70 deletion mutants that are sensitive to perturbations in AFT1 levels using genome-wide synthetic lethal and synthetic dosage lethal screens. Our genetic network reveals that Aft1 affects a diverse range of cellular processes, including the RIM101 pH pathway, cell-wall stability, DNA damage, protein transport, chromosome stability, and mitochondrial function. Surprisingly, only a subset of mutants identified are sensitive to extracellular iron fluctuations or display genetic interactions with mutants of iron regulon genes AFT2 or FET3. We demonstrate that Aft1 works in parallel with the RIM101 pH pathway and the role of Aft1 in DNA damage repair is mediated by iron. In contrast, through both directed studies and microarray transcriptional profiling, we show that the role of Aft1 in chromosome maintenance and benomyl resistance is independent of its iron regulatory role, potentially through a nontranscriptional mechanism.

We report here the first large-scale affinity purification and mass spectrometry (AP-MS) study of chromatin-associated protein, in which over 100 different baits involved in chromatin biology were studied by modified chromatin immunopurification (mChIP)-MS. In particular, focus was placed on poorly studied chromatin binding proteins, such as transcription factors, which have been underrepresented in previous AP-MS studies. mChIP-MS analysis of transcription factors identified dense networks of protein associated with chromatin that were composed of specific transcriptional co-activators, information not accessible through the use of classical AP-MS methods. Finally, we demonstrate that novel protein–protein interactions identified in study by mChIP have functional implications exemplified by the detailed study of both the ubiquitination of the proline isomerase Cpr1 and of histone chaperones involved in the regulation of the HTA1-HTB1 promoter. Our work demonstrates the value of targeted interactome studies, in which affinity purification methods are adapted to the needs of specific baits, as is the case for chromatin binding proteins., The maintenance of cellular fitness requires living organisms to integrate multiple signals into coordinated outputs. Central to this process is the regulation of the expression of the genetic information encoded into DNA. As a result, there are numerous constraints imposed on gene expression. The access to DNA is restricted by the formation of nucleosomes, in which DNA is wrapped around histone octamers to form chromatin wherein the volume of DNA is considerably reduced. As such, nucleosome positioning is critical and must be defined precisely, particularly during transcription (Workman, 2006). Furthermore, nucleosomes can be actively assembled/disassembled by histone chaperones and can be made to ‘slide' along DNA by the actions of chromatin remodelers. Moreover, the histone proteins are heavily regulated at the expression level and by extensive post-translational modifications (PTMs) (Campos and Reinberg, 2009). Histone PTMs have also been shown to help recruit numerous chromatin-associated factors in accordance with the histone code (Strahl and Allis, 2000). Although our understanding of chromatin and its roles has improved, we still have limited knowledge of the chromatin-associated protein complexes and their interactions. The characterization of biological systems and of specific subdomain within them, such as chromatin, remains a difficult task. An efficient approach to gain insight in the function of protein is to define its interactome. The underlying principle of protein interaction mapping is that proteins found to interact must be involved in common processes and localization, i.e., guilt by association. The large-scale mapping of proteins interactions allows to annotate protein of unknown functions, implicate protein of known functions in different processes and derive new hypothesis. This is possible because most proteins do not act in isolation but rather as part of complexes, and thus possess interaction partners that can now be detected with the right tools. AP-MS has emerged as a powerful tool for characterizing protein–protein interactions and biological systems in general (Gingras et al, 2007; Gstaiger and Aebersold, 2009). Recently, we reported the development of a novel affinity purification approach termed mChIP, which was designed to improve the characterization of DNA binding proteins interactome (Lambert et al, 2009). The mChIP method consists of a single affinity purification step, whereby chromatin-associated proteins are isolated from mildly sonicated and gently clarified cellular extracts using magnetic beads coated with antibodies (Lambert et al, 2009; Figure 1A). As such, the mChIP approach maintains chromatin fragments in solution enabling their specific purification, something not previously possible in classical AP-MS methods (Lambert et al, 2009). In this study, we report the utilization of mChIP followed by MS for the characterization of more than 100 proteins and their associated protein networks (Figure 1B). We initially focused on DNA-associated proteins that had been poorly characterized in past AP-MS studies, such as transcription factors. In addition, many histone modifiers, such as lysine acetyl transferases (KAT) and lysine methyl transferases, critical components of chromatin function and regulation, were also studied by mChIP. This resulted in raw non-redundant mChIP-MS data containing ∼9000 protein–protein interactions between ∼900 proteins. Following a two-step curation process designed to remove common contaminants and protein not specifically associated with the baits under study, a high confidence mChIP-MS data set was produced containing 2966 protein–protein interactions between 724 proteins (Figure 1B). It is important to note that our curation strategy was capable of maintaining the majority of the protein–protein interaction identified in previous AP-MS studies, while removing the bulk of protein–protein interaction not related to chromatin biology. Further analysis of the mChIP-MS data set revealed that for most bait tested, mChIP-MS resulted in the identification of more interaction partners than classical TAP-MS. Visualization of the mChIP-MS data set was achieved by generating heat maps from two-dimensional hierarchical clustering of the bait–prey interactions. This revealed numerous clusters within our data set supporting functional relationship. For instance, mChIP analysis of the highly homologous heat-shock-inducible transcription factors Msn2 and Msn4 clustered with different transcriptional co-activators. Importantly, our analysis also revealed key differences in the co-activators associated with Msn2 and Msn4 relevant to their function. Another example that we explore in greater details is the Cpr1 proline isomerase, a known member of the Set3 complex (Pijnappel et al, 2001). mChIP-MS analysis of Cpr1 revealed an extended network of associated proteins, including the E3 ubiquitin ligase Bre1 and its association partner Lge1 (Figure 5A). This association raised the possibility of a direct action of Bre1/Lge1 on Cpr1 to ubiquitinate it. In targeted experiments, we observed that Cpr1 is in fact ubiquitinated in a process involving Bre1/Lge1 (Figure 5E), confirming their functional relationship. As such, mChIP is capable of uncovering novel protein–protein interactions with physiological impacts. In this study, we report how the use of an AP-MS method designed for a given class of protein (chromatin-associated proteins) can help uncover numerous novel protein–protein interactions. Furthermore, our work detected dense chromatin-associated protein networks being co-purified with multiple transcription factors and other DNA binding proteins. The fact that even in the best-characterized model organism Saccharomyces cerevisiae, thousands of novel protein–protein interactions can be detected supports our view that targeted interactome studies are worthwhile and desirable. As such, the budding yeast interactome can still be consider incomplete and warrant further study., We previously reported a novel affinity purification (AP) method termed modified chromatin immunopurification (mChIP), which permits selective enrichment of DNA-bound proteins along with their associated protein network. In this study, we report a large-scale study of the protein network of 102 chromatin-related proteins from budding yeast that were analyzed by mChIP coupled to mass spectrometry. This effort resulted in the detection of 2966 high confidence protein associations with 724 distinct preys. mChIP resulted in significantly improved interaction coverage as compared with classical AP methodology for ∼75% of the baits tested. Furthermore, mChIP successfully identified novel binding partners for many lower abundance transcription factors that previously failed using conventional AP methodologies. mChIP was also used to perform targeted studies, particularly of Asf1 and its associated proteins, to allow for a understanding of the physical interplay between Asf1 and two other histone chaperones, Rtt106 and the HIR complex, to be gained.

The Saccharomyces cerevisiae NuA4 histone acetyltransferase complex catalyzes the acetylation of histone H4 and the histone variant Htz1 to regulate key cellular events, including transcription, DNA repair, and faithful chromosome segregation. To further investigate the cellular processes impacted by NuA4, we exploited the nonessential subunits of the complex to build an extensive NuA4 genetic-interaction network map. The map reveals that NuA4 is a genetic hub whose function buffers a diverse range of cellular processes, many not previously linked to the complex, including Golgi complex-to-vacuole vesicle-mediated transport. Further, we probe the role that nonessential subunits play in NuA4 complex integrity. We find that most nonessential subunits have little impact on NuA4 complex integrity and display between 12 and 42 genetic interactions. In contrast, the deletion of EAF1 causes the collapse of the NuA4 complex and displays 148 genetic interactions. Our study indicates that Eaf1 plays a crucial function in NuA4 complex integrity. Further, we determine that Eaf5 and Eaf7 form a subcomplex, which reflects their similar genetic interaction profiles and phenotypes. Our integrative study demonstrates that genetic interaction maps are valuable in dissecting complex structure and provides insight into why the human NuA4 complex, Tip60, has been associated with a diverse range of pathologies.

The histone H2A variant H2A.Z (Saccharomyces cerevisiae Htz1) plays roles in transcription, DNA repair, chromosome stability, and limiting telomeric silencing. The Swr1-Complex (SWR-C) inserts Htz1 into chromatin and shares several subunits with the NuA4 histone acetyltransferase. Furthermore, mutants of these two complexes share several phenotypes, suggesting they may work together. Here we show that NuA4 acetylates Htz1 Lys 14 (K14) after the histone is assembled into chromatin by the SWR-C. K14 mutants exhibit specific defects in chromosome transmission without affecting transcription, telomeric silencing, or DNA repair. Function-specific modifications may help explain how the same component of chromatin can function in diverse pathways.

Accurate chromosome segregation requires the execution and coordination of many processes during mitosis, including DNA replication, sister chromatid cohesion, and attachment of chromosomes to spindle microtubules via the kinetochore complex. Additional pathways are likely involved because faithful chromosome segregation also requires proteins that are not physically associated with the chromosome. Using kinetochore mutants as a starting point, we have identified genes with roles in chromosome stability by performing genome-wide screens employing synthetic genetic array methodology. Two genetic approaches (a series of synthetic lethal and synthetic dosage lethal screens) isolated 211 nonessential deletion mutants that were unable to tolerate defects in kinetochore function. Although synthetic lethality and synthetic dosage lethality are thought to be based upon similar genetic principles, we found that the majority of interactions associated with these two screens were nonoverlapping. To functionally characterize genes isolated in our screens, a secondary screen was performed to assess defects in chromosome segregation. Genes identified in the secondary screen were enriched for genes with known roles in chromosome segregation. We also uncovered genes with diverse functions, such as RCS1, which encodes an iron transcription factor. RCS1 was one of a small group of genes identified in all three screens, and we used genetic and cell biological assays to confirm that it is required for chromosome stability. Our study shows that systematic genetic screens are a powerful means to discover roles for uncharacterized genes and genes with alternative functions in chromosome maintenance that may not be discovered by using proteomics approaches. © 2005 by The National Academy of Sciences of the USA., link_to_subscribed_fulltext

Methods to systematically test drugs against all possible proteins in a cell are needed to identify the targets underlying their therapeutic action and unwanted effects. Here, we show that a genome-wide drug-induced haploinsufficiency screen by using yeast can reveal drug mode of action in yeast and can be used to predict drug mode of action in human cells. We demonstrate that dihydromotuporamine C, a compound in preclinical development that inhibits angiogenesis and metastasis by an unknown mechanism, targets sphingolipid metabolism. The systematic, unbiased and genome-wide nature of this technique makes it attractive as a general approach to identify cellular pathways affected by drugs.

The budding yeast centromere-kinetochore complex ensures high-fidelity chromosome segregation in mitosis and meiosis by mediating the attachment and movement of chromosomes along spindle microtubules. To identify new genes and pathways whose function impinges on chromosome transmission, we developed a genomic haploinsufficiency modifier screen and used ctf13-30, encoding a mutant core kinetochore protein, as the reference point. We demonstrate through a series of secondary screens that the genomic modifier screen is a successful method for identifying genes that encode nonessential proteins required for the fidelity of chromosome segregation. One gene isolated in our screen was RSC2, a nonessential subunit of the RSC chromatin remodeling complex. rsc2 mutants have defects in both chromosome segregation and cohesion, but the localization of kinetochore proteins to centromeres is not affected. We determined that, in the absence of RSC2, cohesin could still associate with chromosomes but fails to achieve proper cohesion between sister chromatids, indicating that RSC has a role in the establishment of cohesion. In addition, numerous subunits of RSC were affinity purified and a new component of RSC, Rtt102, was identified. Our work indicates that only a subset of the nonessential RSC subunits function in maintaining chromosome transmission fidelity.

The process of gene expression is inherently stochastic and leads to differences in protein abundance from one cell to another. A new study shows that this protein noise is unexpectedly predictable, providing important new insights into the properties and origins of variability in gene expression.