The intrinsically disordered region of coronins fine-tunes oligomerization and actin polymerization.

Coronins play critical roles in actin network formation. The diverse functions of coronins are regulated by the structured N-terminal ß propeller and the C-terminal coiled coil (CC). However, less is known about a middle "unique region" (UR), which is an intrinsically disordered region (IDR). The UR/IDR is an evolutionarily conserved signature in the coronin family. By integrating biochemical and cell biology experiments, coarse-grained simulations, and protein engineering, we find that the IDR optimizes the biochemical activities of coronins in vivo and in vitro. The budding yeast coronin IDR plays essential roles in regulating Crn1 activity by fine-tuning CC oligomerization and maintaining Crn1 as a tetramer. The IDR-guided optimization of Crn1 oligomerization is critical for F-actin cross-linking and regulation of Arp2/3-mediated actin polymerization. The final oligomerization status and homogeneity of Crn1 are contributed by three examined factors: helix packing, the energy landscape of the CC, and the length and molecular grammar of the IDR.

Light Stress in Yeasts: Signaling and Responses in Creatures of the Night.

Living organisms on the surface biosphere are periodically yet consistently exposed to light. The adaptive or protective evolution caused by this source of energy has led to the biological systems present in a large variety of organisms, including fungi. Among fungi, yeasts have developed essential protective responses against the deleterious effects of light. Stress generated by light exposure is propagated through the synthesis of hydrogen peroxide and mediated by regulatory factors that are also involved in the response to other stressors. These have included Msn2/4, Crz1, Yap1, and Mga2, thus suggesting that light stress is a common factor in the yeast environmental response.

Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase.

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. In vitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

A Genome-Wide Screen Reveals That Endocytic Genes Are Important for Pma1p Asymmetry during Cell Division in Saccharomyces cerevisiae.

An asymmetry in cytosolic pH between mother and daughter cells was reported to underlie cellular aging in the budding yeast Saccharomyces cerevisiae; however, the underlying mechanism remains unknown. Preferential accumulation of Pma1p, which pumps cytoplasmic protons out of cells, at the plasma membrane of mother cells, but not of their newly-formed daughter cells, is believed to be responsible for the pH increase in mother cells by reducing the level of cytoplasmic protons. This, in turn, decreases the acidity of vacuoles, which is well correlated with aging of yeast cells. In this study, to identify genes that regulate the preferential accumulation of Pma1p in mother cells, we performed a genome-wide screen using a collection of single gene deletion yeast strains. A subset of genes involved in the endocytic pathway, such as VPS8, VPS9, and VPS21, was important for Pma1p accumulation. Unexpectedly, however, there was little correlation between deletion of each of these genes and the replicative lifespan of yeast, suggesting that Pma1p accumulation in mother cells is not the key determinant that underlies aging of mother cells.

Disruption of YCP4 enhances freeze-thaw tolerance in Saccharomyces cerevisiae.

OBJECTIVE: This study aimed to identify genes related to freeze-thaw tolerance and elucidate the tolerance mechanism in yeast Saccharomyces cerevisiae as an appropriate eukaryote model. RESULTS: In this study, one tolerant strain exposed to freeze-thaw stress was isolated by screening a transposon-mediated mutant library and the disrupted gene was identified to be YCP4. In addition, this phenotype related to freeze-thaw tolerance was confirmed by deletion and overexpressing of this corresponding gene. This mutant strain showed a freeze-thaw tolerance by reducing the intracellular level of reactive oxygen species and the activation of the MSN2/4 and STRE-mediated genes such as CTT1 and HSP12. CONCLUSIONS: Disruption of YCP4 in S. cerevisiae results in increased tolerance to freeze-thaw stress.

A positive feedback loop involving the Spa2 SHD domain contributes to focal polarization.

Focal polarization is necessary for finely arranged cell-cell interactions. The yeast mating projection, with its punctate polarisome, is a good model system for this process. We explored the critical role of the polarisome scaffold protein Spa2 during yeast mating with a hypothesis motivated by mathematical modeling and tested by in vivo experiments. Our simulations predicted that two positive feedback loops generate focal polarization, including a novel feedback pathway involving the N-terminal domain of Spa2. We characterized the latter using loss-of-function and gain-of-function mutants. The N-terminal region contains a Spa2 Homology Domain (SHD) which is conserved from yeast to humans, and when mutated largely reproduced the spa2Δ phenotype. Our work together with published data show that the SHD domain recruits Msb3/4 that stimulates Sec4-mediated transport of Bud6 to the polarisome. There, Bud6 activates Bni1-catalyzed actin cable formation, recruiting more Spa2 and completing the positive feedback loop. We demonstrate that disrupting this loop at any point results in morphological defects. Gain-of-function perturbations partially restored focal polarization in a spa2 loss-of-function mutant without restoring localization of upstream components, thus supporting the pathway order. Thus, we have collected data consistent with a novel positive feedback loop that contributes to focal polarization during pheromone-induced polarization in yeast.

Site-specific Phosphorylation of Histone H3K36 Methyltransferase Set2p and Demethylase Jhd1p is Required for Stress Responses in Saccharomyces cerevisiae.

Histone lysine methylation is a key epigenetic modification that regulates eukaryotic transcription. In Saccharomyces cerevisiae, it is controlled by a reduced but evolutionarily conserved suite of methyltransferase (Set1p, Set2p, Dot1p, and Set5p) and demethylase (Jhd1p, Jhd2p, Rph1p, and Gis1p) enzymes. Many of these enzymes are extensively phosphorylated in vivo; however, the functions of almost all phosphosites remain unknown. Here, we comprehensively analyse the phosphoregulation of the yeast histone methylation network by functionally investigating 40 phosphosites on six enzymes. A total of 82 genomically-edited S. cerevisiae strains were generated through mutagenesis of sites to aspartate as a phosphomimetic or alanine as a phosphonull. These phosphosite mutants were screened for changes in native H3K4, H3K36, and H3K79 methylation levels, and for sensitivity to environmental stress conditions. For methyltransferase Set2p, we found that phosphorylation at threonine 127 significantly decreased H3K36 methylation in vivo, and that an N-terminal phosphorylation cluster at serine residues 6, 8, and 10 is required for the diamide stress response. Proteomic analysis of Set2p phosphosite mutants revealed a specific downregulation of membrane-associated proteins and processes, consistent with changes brought about by SET2 deletion and the sensitivity of mutants to diamide. For demethylase Jhd1p, we found that its sole phosphorylation site at serine 44 is required for the cold stress response. This study represents the first systematic investigation into the phosphoregulation of the epigenetic network in any eukaryote, and shows that phosphosites on histone methylation enzymes are required for a normal cellular response to stress in S.cerevisiae.

Rad54 and Rdh54 prevent Srs2-mediated disruption of Rad51 presynaptic filaments.

Srs2 is a superfamily 1 (SF1) helicase that participates in several pathways necessary for the repair of damaged DNA. Srs2 regulates formation of early homologous recombination (HR) intermediates by actively removing the recombinase Rad51 from single-stranded DNA (ssDNA). It is not known whether and how Srs2 itself is down-regulated to allow for timely HR progression. Rad54 and Rdh54 are two closely related superfamily 2 (SF2) motor proteins that promote the formation of Rad51-dependent recombination intermediates. Rad54 and Rdh54 bind tightly to Rad51-ssDNA and act downstream of Srs2, suggesting that they may affect the ability of Srs2 to dismantle Rad51 filaments. Here, we used DNA curtains to determine whether Rad54 and Rdh54 alter the ability of Srs2 to disrupt Rad51 filaments. We show that Rad54 and Rdh54 act synergistically to greatly restrict the antirecombinase activity of Srs2. Our findings suggest that Srs2 may be accorded only a limited time window to act and that Rad54 and Rdh54 fulfill a role of prorecombinogenic licensing factors.

Hierarchical integration of mitochondrial and nuclear positioning pathways by the Num1 EF hand.

Positioning organelles at the right place and time is critical for their function and inheritance. In budding yeast, mitochondrial and nuclear positioning require the anchoring of mitochondria and dynein to the cell cortex by clusters of Num1. We have previously shown that mitochondria drive the assembly of cortical Num1 clusters, which then serve as anchoring sites for mitochondria and dynein. When mitochondrial inheritance is inhibited, mitochondrial-driven assembly of Num1 in buds is disrupted and defects in dynein-mediated spindle positioning are observed. Using a structure-function approach to dissect the mechanism of mitochondria-dependent dynein anchoring, we found that the EF hand-like motif (EFLM) of Num1 and its ability to bind calcium are required to bias dynein anchoring on mitochondria-associated Num1 clusters. Consistently, when the EFLM is disrupted, we no longer observe defects in dynein activity following inhibition of mitochondrial inheritance. Thus, the Num1 EFLM functions to bias dynein anchoring and activity in nuclear inheritance subsequent to mitochondrial inheritance. We hypothesize that this hierarchical integration of organelle positioning pathways by the Num1 EFLM contributes to the regulated order of organelle inheritance during the cell cycle.

Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae.

The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: the recruitment of Sir proteins to silencers and their spread throughout the silenced domain. We developed a method to study these two processes at single basepair resolution. Using a fusion protein between the heterochromatin protein Sir3 and the nonsite-specific bacterial adenine methyltransferase M.EcoGII, we mapped sites of Sir3-chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein and by ChIP-seq to map the distribution of Sir3-M.EcoGII. A silencing-deficient mutant of Sir3 lacking its Bromo-Adjacent Homology (BAH) domain, sir3-bah∆, was still recruited to HML, HMR, and telomeres. However, in the absence of the BAH domain, it was unable to spread away from those recruitment sites. Overexpression of Sir3 did not lead to further spreading at HML, HMR, and most telomeres. A few exceptional telomeres, like 6R, exhibited a small amount of Sir3 spreading, suggesting that boundaries at telomeres responded variably to Sir3-M.EcoGII overexpression. Finally, by using a temperature-sensitive allele of SIR3 fused to M.ECOGII, we tracked the positions first methylated after induction and found that repression of genes at HML and HMR began before Sir3 occupied the entire locus.

The mitospecific domain of Mrp7 (bL27) supports mitochondrial translation during fermentation and is required for effective adaptation to respiration.

We demonstrate here that mitoribosomal protein synthesis, responsible for the synthesis of oxidative phosphorylation (OXPHOS) subunits encoded by the mitochondrial genome, occurs at high levels during glycolysis fermentation and in a manner uncoupled from OXPHOS complex assembly regulation. Furthermore, we provide evidence that the mitospecific domain of Mrp7 (bL27), a mitoribosomal component, is required to maintain mitochondrial protein synthesis during fermentation but is not required under respiration growth conditions. Maintaining mitotranslation under high-glucose-fermentation conditions also involves Mam33 (p32/gC1qR homologue), a binding partner of Mrp7's mitospecific domain, and together they confer a competitive advantage for a cell's ability to adapt to respiration-based metabolism when glucose becomes limiting. Furthermore, our findings support that the mitoribosome, and specifically the central protuberance region, may be differentially regulated and/or assembled, under the different metabolic conditions of fermentation and respiration. On the basis of our findings, we propose that the purpose of mitotranslation is not limited to the assembly of OXPHOS complexes, but also plays a role in mitochondrial signaling critical for switching cellular metabolism from a glycolysis- to a respiration-based state.

Role and Regulation of the RECQL4 Family during Genomic Integrity Maintenance.

RECQL4 is a member of the evolutionarily conserved RecQ family of 3' to 5' DNA helicases. RECQL4 is critical for maintaining genomic stability through its functions in DNA repair, recombination, and replication. Unlike many DNA repair proteins, RECQL4 has unique functions in many of the central DNA repair pathways such as replication, telomere, double-strand break repair, base excision repair, mitochondrial maintenance, nucleotide excision repair, and crosslink repair. Consistent with these diverse roles, mutations in RECQL4 are associated with three distinct genetic diseases, which are characterized by developmental defects and/or cancer predisposition. In this review, we provide an overview of the roles and regulation of RECQL4 during maintenance of genome homeostasis.

VID22 counteracts G-quadruplex-induced genome instability.

Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair. vid22Δ cells exhibit increased levels of endogenous DNA damage, chronic DNA damage response activation and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. Vid22 binds to and protects DNA at G4-containing regions both in vitro and in vivo. Loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. Moreover, the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism.

Function and molecular mechanism of N-terminal acetylation in autophagy.

Acetyl ligation to the amino acids in a protein is an important posttranslational modification. However, in contrast to lysine acetylation, N-terminal acetylation is elusive in terms of its cellular functions. Here, we identify Nat3 as an N-terminal acetyltransferase essential for autophagy, a catabolic pathway for bulk transport and degradation of cytoplasmic components. We identify the actin cytoskeleton constituent Act1 and dynamin-like GTPase Vps1 (vacuolar protein sorting 1) as substrates for Nat3-mediated N-terminal acetylation of the first methionine. Acetylated Act1 forms actin filaments and therefore promotes the transport of Atg9 vesicles for autophagosome formation; acetylated Vps1 recruits and facilitates bundling of the SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor) complex for autophagosome fusion with vacuoles. Abolishment of the N-terminal acetylation of Act1 and Vps1 is associated with blockage of upstream and downstream steps of the autophagy process. Therefore, our work shows that protein N-terminal acetylation plays a critical role in controlling autophagy by fine-tuning multiple steps in the process.

The chromatin remodeler Ino80 mediates RNAPII pausing site determination.

BACKGROUND: Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step for the precise regulation of gene expression. Despite the apparent close relationship between promoter-proximal pausing and nucleosome, the role of chromatin remodeler governing this step has mainly remained elusive. RESULTS: Here, we report highly confined RNAPII enrichments downstream of the transcriptional start site in Saccharomyces cerevisiae using PRO-seq experiments. This non-uniform distribution of RNAPII exhibits both similar and different characteristics with promoter-proximal pausing in Schizosaccharomyces pombe and metazoans. Interestingly, we find that Ino80p knockdown causes a significant upstream transition of promoter-proximal RNAPII for a subset of genes, relocating RNAPII from the main pausing site to the alternative pausing site. The proper positioning of RNAPII is largely dependent on nucleosome context. We reveal that the alternative pausing site is closely associated with the + 1 nucleosome, and nucleosome architecture around the main pausing site of these genes is highly phased. In addition, Ino80p knockdown results in an increase in fuzziness and a decrease in stability of the + 1 nucleosome. Furthermore, the loss of INO80 also leads to the shift of promoter-proximal RNAPII toward the alternative pausing site in mouse embryonic stem cells. CONCLUSIONS: Based on our collective results, we hypothesize that the highly conserved chromatin remodeler Ino80p is essential in establishing intact RNAPII pausing during early transcription elongation in various organisms, from budding yeast to mouse.

The Role of the Rad55-Rad57 Complex in DNA Repair.

Homologous recombination (HR) is a mechanism conserved from bacteria to humans essential for the accurate repair of DNA double-stranded breaks, and maintenance of genome integrity. In eukaryotes, the key DNA transactions in HR are catalyzed by the Rad51 recombinase, assisted by a host of regulatory factors including mediators such as Rad52 and Rad51 paralogs. Rad51 paralogs play a crucial role in regulating proper levels of HR, and mutations in the human counterparts have been associated with diseases such as cancer and Fanconi Anemia. In this review, we focus on the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57, which has served as a model for understanding the conserved role of Rad51 paralogs in higher eukaryotes. Here, we discuss the results from early genetic studies, biochemical assays, and new single-molecule observations that have together contributed to our current understanding of the molecular role of Rad55-Rad57 in HR.

Isoflurane induces Art2-Rsp5-dependent endocytosis of Bap2 in yeast.

Although general anesthesia is indispensable during modern surgical procedures, the mechanism by which inhalation anesthetics act on the synaptic membrane at the molecular and cellular level is largely unknown. In this study, we used yeast cells to examine the effect of isoflurane, an inhalation anesthetic, on membrane proteins. Bap2, an amino acid transporter localized on the plasma membrane, was endocytosed when yeast cells were treated with isoflurane. Depletion of RSP5, an E3 ligase, prevented this endocytosis and Bap2 was ubiquitinated in response to isoflurane, indicating an ubiquitin-dependent process. Screening all the Rsp5 binding adaptors showed that Art2 plays a central role in this process. These results suggest that isoflurane affects Bap2 via an Art2-Rsp5-dependent ubiquitination system.

Characterization of nuclear pore complex targeting domains in Pom152 in Saccharomyces cerevisiae.

Pom152 is a transmembrane protein within the nuclear pore complex (NPC) of fungi that is important for NPC assembly and structure. Pom152 is comprised of a short amino-terminal region that remains on the cytosolic side of the nuclear envelope (NE) and interacts with NPC proteins, a transmembrane domain, and a large, glycosylated carboxy-terminal domain within the NE lumen. Here we show that the N-terminal 200 amino acids of Pom152 that include only the amino-terminal and transmembrane regions are sufficient for localization to the NPC. Full-length, glycosylation-deficient, and truncated Pom152-GFP chimeras expressed in cells containing endogenous Pom152 localize to both NPCs and cortical endoplasmic reticulum (ER). Expression of Pom152-GFP fusions in pom152Δ cells results in detectable localization at only the NE by full-length and amino-terminal Pom152-GFP fusions, but continued retention at both the NE and ER for a chimera lacking just the carboxy-terminal 377 amino acids. Neither deletion of Pom152 nor its carboxy-terminal glycosylation sites altered the nuclear protein export rate of an Msn5/Kap142 protein cargo. These data narrow the Pom152 region sufficient for NPC localization and provide evidence that alterations in other domains may impact Pom152 targeting or affinity for the NPC.

Saccharomyces cerevisiae Fpr1 functions as a chaperone to inhibit protein aggregation.

Peptidyl prolyl isomerases (PPIases) accelerate the rate limiting step of protein folding by catalyzing cis/trans isomerization of peptidyl prolyl bonds. The larger PPIases have been shown to be multi-domain proteins, with functions other than isomerization of the proline-containing peptide bond. Recently, a few smaller PPIases have also been described for their ability to stabilize folding intermediates. The yeast Fpr1 (FK506-sensitive proline rotamase) is a homologue of the mammalian prolyl isomerase FKBP12 (FK506-binding protein of 12 kDa). Its ability to stabilize stressed cellular proteins has not been reported yet. We had earlier reported upregulation of Fpr1 in yeast cells exposed to proteotoxic stress conditions. In this work, we show that yeast Fpr1 exhibits characteristics typical of a general chaperone of the proteostasis network. Aggregation of mutant huntingtin fragment was higher in Fpr1-deleted as compared to parental yeast cells. Overexpression of Fpr1 led to reduced protein aggregation by decreasing the amount of oligomers and diverting the aggregation pathway towards the formation of detergent-soluble species. This correlated well with higher survival of these cells. Purified and enzymatically active yeast Fpr1 was able to inhibit aggregation of mutant huntingtin fragment and luciferase in vitro in a concentration-dependent manner; suggesting a direct action for aggregation inhibitory action of Fpr1. Overexpression of yeast Fpr1 was able to protect E. coli cells against thermal shock. This work establishes the role of Fpr1 in the protein folding network and will be used for the identification of novel pharmacological leads in disease conditions.

Slk19 enhances cross-linking of microtubules by Ase1 and Stu1.

The Saccharomyces cerevisiae protein Slk19 has been shown to localize to kinetochores throughout mitosis and to the spindle midzone in anaphase. However, Slk19 clearly also has an important role for spindle formation and stabilization in prometaphase and metaphase, albeit this role is unresolved. Here we show that Slk19's localization to metaphase spindles in vivo and to microtubules (MTs) in vitro depends on the MT cross-linking protein Ase1 and the MT cross-linking and stabilizing protein Stu1. By analyzing a slk19 mutant that specifically fails to localize to spindles and MTs, we surprisingly found that the presence of Slk19 amplified the amount of Ase1 strongly and that of Stu1 moderately at the metaphase spindle in vivo and at MTs in vitro. Furthermore, Slk19 markedly enhanced the cross-linking of MTs in vitro when added together with Ase1 or Stu1. We therefore suggest that Slk19 recruits additional Ase1 and Stu1 to the interpolar MTs (ipMTs) of metaphase spindles and thus increases their cross-linking and stabilization. This is in agreement with our observation that cells with defective Slk19 localization exhibit shorter metaphase spindles, an increased number of unaligned nuclear MTs, and most likely reduced ipMT overlaps.