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1.
Microbiol Resour Announc ; 10(3)2021 Jan 21.
Article in English | MEDLINE | ID: mdl-33479002

ABSTRACT

We characterized the complete genome sequence of Siphoviridae bacteriophage Erla, an obligatory lytic subcluster EA1 bacteriophage infecting Microbacterium foliorum NRRL B-24224, with a capsid width of 65 nm and a tail length of 112 nm. The 41.5-kb genome, encompassing 62 predicted protein-coding genes, is highly similar (99.52% identity) to that of bacteriophage Calix.

2.
J Cell Sci ; 133(14)2020 07 29.
Article in English | MEDLINE | ID: mdl-32591482

ABSTRACT

PP2ACdc55 (the form of protein phosphatase 2A containing Cdc55) regulates cell cycle progression by reversing cyclin-dependent kinase (CDK)- and polo-like kinase (Cdc5)-dependent phosphorylation events. In S. cerevisiae, Cdk1 phosphorylates securin (Pds1), which facilitates Pds1 binding and inhibits separase (Esp1). During anaphase, Esp1 cleaves the cohesin subunit Scc1 and promotes spindle elongation. Here, we show that PP2ACdc55 directly dephosphorylates Pds1 both in vivo and in vitro Pds1 hyperphosphorylation in a cdc55 deletion mutant enhanced the Pds1-Esp1 interaction, which played a positive role in Pds1 nuclear accumulation and in spindle elongation. We also show that nuclear PP2ACdc55 plays a role during replication stress to inhibit spindle elongation. This pathway acted independently of the known Mec1, Swe1 or spindle assembly checkpoint (SAC) checkpoint pathways. We propose a model where Pds1 dephosphorylation by PP2ACdc55 disrupts the Pds1-Esp1 protein interaction and inhibits Pds1 nuclear accumulation, which prevents spindle elongation, a process that is elevated during replication stress.


Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosome Segregation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphorylation , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Securin , Separase , Spindle Apparatus/metabolism
3.
PLoS Genet ; 14(3): e1007029, 2018 03.
Article in English | MEDLINE | ID: mdl-29561844

ABSTRACT

Anaphase onset is an irreversible cell cycle transition that is triggered by the activation of the protease Separase. Separase cleaves the Mcd1 (also known as Scc1) subunit of Cohesin, a complex of proteins that physically links sister chromatids, triggering sister chromatid separation. Separase is regulated by the degradation of the anaphase inhibitor Securin which liberates Separase from inhibitory Securin/Separase complexes. In many organisms, Securin is not essential suggesting that Separase is regulated by additional mechanisms. In this work, we show that in budding yeast Cdk1 activates Separase (Esp1 in yeast) through phosphorylation to trigger anaphase onset. Esp1 activation is opposed by protein phosphatase 2A associated with its regulatory subunit Cdc55 (PP2ACdc55) and the spindle protein Slk19. Premature anaphase spindle elongation occurs when Securin (Pds1 in yeast) is inducibly degraded in cells that also contain phospho-mimetic mutations in ESP1, or deletion of CDC55 or SLK19. This striking phenotype is accompanied by advanced degradation of Mcd1, disruption of pericentric Cohesin organization and chromosome mis-segregation. Our findings suggest that PP2ACdc55 and Slk19 function redundantly with Pds1 to inhibit Esp1 within pericentric chromatin, and both Pds1 degradation and Cdk1-dependent phosphorylation of Esp1 act together to trigger anaphase onset.


Subject(s)
Anaphase/physiology , CDC2 Protein Kinase/metabolism , Microtubule-Associated Proteins/metabolism , Protein Phosphatase 2/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Separase/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Mutation , Phosphorylation , Protein Phosphatase 2/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Securin/genetics , Securin/metabolism , Separase/genetics , Spindle Apparatus/genetics , Cohesins
4.
Cell Rep ; 22(13): 3427-3439, 2018 03 27.
Article in English | MEDLINE | ID: mdl-29590613

ABSTRACT

Polyphosphates (polyP) are chains of inorganic phosphates found in all cells. Previous work has implicated these chains in diverse functions, but the mechanism of action is unclear. A recent study reports that polyP can be non-enzymatically and covalently attached to lysine residues on yeast proteins Nsr1 and Top1. One question emerging from this work is whether so-called "polyphosphorylation" is unique to these proteins or instead functions as a global regulator akin to other lysine post-translational modifications. Here, we present the results of a screen for polyphosphorylated proteins in yeast. We uncovered 15 targets including a conserved network of proteins functioning in ribosome biogenesis. Multiple genes contribute to polyphosphorylation of targets by regulating polyP synthesis, and disruption of this synthesis results in translation defects as measured by polysome profiling. Finally, we identify 6 human proteins that can be modified by polyP, highlighting the therapeutic potential of manipulating polyphosphorylation in vivo.


Subject(s)
Lysine/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Humans , Organelle Biogenesis , Phosphorylation
5.
G3 (Bethesda) ; 7(4): 1117-1126, 2017 04 03.
Article in English | MEDLINE | ID: mdl-28188183

ABSTRACT

Heterochromatin formation in the yeast Saccharomyces cerevisiae is characterized by the assembly of the Silent Information Regulator (SIR) complex, which consists of the histone deacetylase Sir2 and the structural components Sir3 and Sir4, and binds to unmodified nucleosomes to provide gene silencing. Sir3 contains an AAA+ ATPase-like domain, and mutations in an exposed loop on the surface of this domain abrogate Sir3 silencing function in vivo, as well in vitro binding to the Sir2/Sir4 subcomplex. Here, we found that the removal of a single methyl group in the C-terminal coiled-coil domain (mutation T1314S) of Sir4 was sufficient to restore silencing at the silent mating-type loci HMR and HML to a Sir3 version with a mutation in this loop. Restoration of telomeric silencing required further mutations of Sir4 (E1310V and K1325R). Significantly, these mutations in Sir4 restored in vitro complex formation between Sir3 and the Sir4 coiled-coil, indicating that the improved affinity between Sir3 and Sir4 is responsible for the restoration of silencing. Altogether, these observations highlight remarkable properties of selected amino-acid changes at the Sir3-Sir4 interface that modulate the affinity of the two proteins.


Subject(s)
Heterochromatin/metabolism , Mutant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Silent Information Regulator Proteins, Saccharomyces cerevisiae/chemistry , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Gene Silencing , Genetic Loci , Mutant Proteins/chemistry , Mutation/genetics , Protein Binding , Protein Domains , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Structure-Activity Relationship , Suppression, Genetic , Telomere/metabolism
6.
Mol Cell ; 64(4): 720-733, 2016 11 17.
Article in English | MEDLINE | ID: mdl-27818142

ABSTRACT

Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to coordinate RPG and rRNA transcription.


Subject(s)
Gene Expression Regulation, Fungal , RNA, Ribosomal/genetics , Ribosomal Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Trans-Activators/genetics , Organelle Biogenesis , Promoter Regions, Genetic , Protein Binding , RNA Polymerase I/genetics , RNA Polymerase I/metabolism , RNA, Ribosomal/biosynthesis , Ribosomal Proteins/biosynthesis , Ribosomal Proteins/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Trans-Activators/metabolism , Transcription, Genetic
7.
Sci Rep ; 6: 27697, 2016 Jun 21.
Article in English | MEDLINE | ID: mdl-27323850

ABSTRACT

Conditional gene expression systems that enable inducible and reversible transcriptional control are essential research tools and have broad applications in biomedicine and biotechnology. The reverse tetracycline transcriptional activator is a canonical system for engineered gene expression control that enables graded and gratuitous modulation of target gene transcription in eukaryotes from yeast to human cell lines and transgenic animals. However, the system has a tendency to activate transcription even in the absence of tetracycline and this leaky target gene expression impedes its use. Here, we identify single amino-acid substitutions that greatly enhance the dynamic range of the system in yeast by reducing leaky transcription to undetectable levels while retaining high expression capacity in the presence of inducer. While the mutations increase the inducer concentration required for full induction, additional sensitivity-enhancing mutations can compensate for this effect and confer a high degree of robustness to the system. The novel transactivator variants will be useful in applications where tight and tunable regulation of gene expression is paramount.


Subject(s)
Biotechnology/methods , Tetracycline/metabolism , Trans-Activators/genetics , Transcriptional Activation/genetics , Amino Acid Substitution/genetics , Animals , Animals, Genetically Modified , Cell Line , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Humans , Tetracycline/pharmacology , Trans-Activators/metabolism , Trans-Activators/pharmacology , Transcriptional Activation/drug effects , Yeasts/genetics
8.
Genetics ; 202(3): 903-10, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26715668

ABSTRACT

Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase.


Subject(s)
CDC2 Protein Kinase/metabolism , Protein Tyrosine Phosphatases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Tyrosine/chemistry , CDC2 Protein Kinase/genetics , Phosphorylation , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Protein Tyrosine Phosphatases/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , ras-GRF1/genetics , ras-GRF1/metabolism
9.
PLoS Genet ; 11(11): e1005425, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26587833

ABSTRACT

Changes in the locations and boundaries of heterochromatin are critical during development, and de novo assembly of silent chromatin in budding yeast is a well-studied model for how new sites of heterochromatin assemble. De novo assembly cannot occur in the G1 phase of the cell cycle and one to two divisions are needed for complete silent chromatin assembly and transcriptional repression. Mutation of DOT1, the histone H3 lysine 79 (K79) methyltransferase, and SET1, the histone H3 lysine 4 (K4) methyltransferase, speed de novo assembly. These observations have led to the model that regulated demethylation of histones may be a mechanism for how cells control the establishment of heterochromatin. We find that the abundance of Sir4, a protein required for the assembly of silent chromatin, decreases dramatically during a G1 arrest and therefore tested if changing the levels of Sir4 would also alter the speed of de novo establishment. Halving the level of Sir4 slows heterochromatin establishment, while increasing Sir4 speeds establishment. yku70Δ and ubp10Δ cells also speed de novo assembly, and like dot1Δ cells have defects in subtelomeric silencing, suggesting that these mutants may indirectly speed de novo establishment by liberating Sir4 from telomeres. Deleting RIF1 and RIF2, which suppresses the subtelomeric silencing defects in these mutants, rescues the advanced de novo establishment in yku70Δ and ubp10Δ cells, but not in dot1Δ cells, suggesting that YKU70 and UBP10 regulate Sir4 availability by modulating subtelomeric silencing, while DOT1 functions directly to regulate establishment. Our data support a model whereby the demethylation of histone H3 K79 and changes in Sir4 abundance and availability define two rate-limiting steps that regulate de novo assembly of heterochromatin.


Subject(s)
Gene Silencing , Heterochromatin/genetics , Saccharomyces cerevisiae/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/physiology , DNA-Binding Proteins/genetics , Epistasis, Genetic , G1 Phase , Gene Deletion , Mutation , Nuclear Proteins/genetics , Repressor Proteins/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/genetics , Telomere , Telomere-Binding Proteins/genetics , Ubiquitin Thiolesterase/genetics
10.
Exp Cell Res ; 330(2): 248-266, 2015 Jan 15.
Article in English | MEDLINE | ID: mdl-25445790

ABSTRACT

In cell culture, many adherent mammalian cells undergo substantial actin cytoskeleton rearrangement prior to mitosis as they detach from the extracellular matrix and become spherical. At the end of mitosis, the actin cytoskeleton is required for cytokinesis and the reassembly of interphase structures as cells spread and reattach to substrate. To understand the processes regulating mitotic cytoskeletal remodeling, we studied how mitotic phosphorylation regulates filamin A (FLNa). FLNa is an actin-crosslinking protein that was previously identified as a cyclin-dependent kinase 1 (Cdk1) binding partner and substrate in vitro. Using quantitative label-based mass spectrometry, we find that FLNa serines 1084, 1459 and 1533 are phosphorylated in mitotic HeLa cells and all three sites match the phosphorylation consensus sequence of Cdk1. To investigate the functional role of mitotic FLNa phosphorylation, we mutated serines 1084, 1459 and 1533 to nonphosphorylatable alanine residues and expressed GFP-tagged FLNa(S1084A,S1459A,S1533A) (FLNa-AAA GFP) in a FLNa-deficient human melanoma cell line called M2. M2 cells expressing FLNa-AAA GFP have enhanced FLNa-AAA GFP and actin localization at sites of contact between daughter cells, impaired post-mitotic daughter cell separation and defects in cell migration. Therefore, mitotic phosphorylation of FLNa is important for successful cell division and interphase cell behavior.


Subject(s)
Cyclin B1/metabolism , Cyclin-Dependent Kinases/metabolism , Filamins/metabolism , Mitosis/physiology , Actin Cytoskeleton/physiology , Actins/metabolism , CDC2 Protein Kinase , Cell Line, Tumor , Cell Movement/genetics , Cell Movement/physiology , Cytokinesis/physiology , Filamins/genetics , Green Fluorescent Proteins/genetics , HeLa Cells , Humans , Melanoma , Mutation , Phosphorylation , Pseudopodia/physiology
11.
Blood ; 124(18): 2867-71, 2014 Oct 30.
Article in English | MEDLINE | ID: mdl-25193871

ABSTRACT

Mutations in genes encoding proteins that are involved in mitochondrial heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis have previously been implicated in the pathogenesis of the congenital sideroblastic anemias (CSAs). We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Here we demonstrate that SIFD is caused by biallelic mutations in TRNT1, the gene encoding the CCA-adding enzyme essential for maturation of both nuclear and mitochondrial transfer RNAs. Using budding yeast lacking the TRNT1 homolog, CCA1, we confirm that the patient-associated TRNT1 mutations result in partial loss of function of TRNT1 and lead to metabolic defects in both the mitochondria and cytosol, which can account for the phenotypic pleiotropy.


Subject(s)
Anemia, Sideroblastic/congenital , Anemia, Sideroblastic/genetics , Developmental Disabilities/complications , Fever/complications , Genetic Diseases, X-Linked/genetics , Immunologic Deficiency Syndromes/complications , Mutation/genetics , RNA Nucleotidyltransferases/genetics , Alleles , Anemia, Sideroblastic/complications , Anemia, Sideroblastic/enzymology , Developmental Disabilities/genetics , Fever/genetics , Genetic Diseases, X-Linked/complications , Genetic Diseases, X-Linked/enzymology , HEK293 Cells , Humans , Immunologic Deficiency Syndromes/genetics
12.
J Biol Chem ; 289(19): 13186-96, 2014 May 09.
Article in English | MEDLINE | ID: mdl-24648511

ABSTRACT

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) is a modification of new H3 molecules deposited throughout the genome during S-phase. H3K56ac is removed by the sirtuins Hst3 and Hst4 at later stages of the cell cycle. Previous studies indicated that regulated degradation of Hst3 plays an important role in the genome-wide waves of H3K56 acetylation and deacetylation that occur during each cell cycle. However, little is known regarding the mechanism of cell cycle-regulated Hst3 degradation. Here, we demonstrate that Hst3 instability in vivo is dependent upon the ubiquitin ligase SCF(Cdc4) and that Hst3 is phosphorylated at two Cdk1 sites, threonine 380 and threonine 384. This creates a diphosphorylated degron that is necessary for Hst3 polyubiquitylation by SCF(Cdc4). Mutation of the Hst3 diphospho-degron does not completely stabilize Hst3 in vivo, but it nonetheless results in a significant fitness defect that is particularly severe in mutant cells treated with the alkylating agent methyl methanesulfonate. Unexpectedly, we show that Hst3 can be degraded between G2 and anaphase, a window of the cell cycle where Hst3 normally mediates genome-wide deacetylation of H3K56. Our results suggest an intricate coordination between Hst3 synthesis, genome-wide H3K56 deacetylation by Hst3, and cell cycle-regulated degradation of Hst3 by cyclin-dependent kinases and SCF(Cdc4).


Subject(s)
Cell Cycle Proteins/metabolism , Cell Cycle/physiology , F-Box Proteins/metabolism , Genome, Fungal/physiology , Histone Deacetylases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/physiology , Acetylation , Cell Cycle Proteins/genetics , Enzyme Stability/physiology , F-Box Proteins/genetics , Histone Deacetylases/genetics , Histones/genetics , Histones/metabolism , Phosphorylation/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin-Protein Ligases/genetics
13.
J Cell Biol ; 201(6): 843-62, 2013 Jun 10.
Article in English | MEDLINE | ID: mdl-23751495

ABSTRACT

Cdk1 drives both mitotic entry and the metaphase-to-anaphase transition. Past work has shown that Wee1 inhibition of Cdk1 blocks mitotic entry. Here we show that the budding yeast Wee1 kinase, Swe1, also restrains the metaphase-to-anaphase transition by preventing Cdk1 phosphorylation and activation of the mitotic form of the anaphase-promoting complex/cyclosome (APC(Cdc20)). Deletion of SWE1 or its opposing phosphatase MIH1 (the budding yeast cdc25(+)) altered the timing of anaphase onset, and activation of the Swe1-dependent morphogenesis checkpoint or overexpression of Swe1 blocked cells in metaphase with reduced APC activity in vivo and in vitro. The morphogenesis checkpoint also depended on Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A). cdc55Δ checkpoint defects were rescued by mutating 12 Cdk1 phosphorylation sites on the APC, demonstrating that the APC is a target of this checkpoint. These data suggest a model in which stepwise activation of Cdk1 and inhibition of PP2A(Cdc55) triggers anaphase onset.


Subject(s)
Anaphase/physiology , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Metaphase/physiology , Protein-Tyrosine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , CDC2 Protein Kinase/genetics , Cell Cycle Proteins/genetics , Genes, cdc/physiology , Phosphorylation/physiology , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , Protein-Tyrosine Kinases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Ubiquitination/physiology , ras-GRF1/genetics , ras-GRF1/metabolism
14.
Mol Cell Proteomics ; 8(4): 870-82, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19106085

ABSTRACT

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.


Subject(s)
Chromatin/metabolism , Nuclear Proteins/analysis , Proteomics/methods , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae/metabolism , Chromatin Immunoprecipitation , DNA, Fungal/metabolism , Histones/metabolism , Nuclear Proteins/isolation & purification , Protein Binding , Saccharomyces cerevisiae Proteins/isolation & purification
15.
Mol Cell Biol ; 28(22): 6903-18, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18794362

ABSTRACT

Silent chromatin in Saccharomyces cerevisiae is established in a stepwise process involving the SIR complex, comprised of the histone deacetylase Sir2 and the structural components Sir3 and Sir4. The Sir3 protein, which is the primary histone-binding component of the SIR complex, forms oligomers in vitro and has been proposed to mediate the spreading of the SIR complex along the chromatin fiber. In order to analyze the role of Sir3 in the spreading of the SIR complex, we performed a targeted genetic screen for alleles of SIR3 that dominantly disrupt silencing. Most mutations mapped to a single surface in the conserved N-terminal BAH domain, while one, L738P, localized to the AAA ATPase-like domain within the C-terminal half of Sir3. The BAH point mutants, but not the L738P mutant, disrupted the interaction between Sir3 and nucleosomes. In contrast, Sir3-L738P bound the N-terminal tail of histone H4 more strongly than wild-type Sir3, indicating that misregulation of the Sir3 C-terminal histone-binding activity also disrupted spreading. Our results underscore the importance of proper interactions between Sir3 and the nucleosome in silent chromatin assembly. We propose a model for the spreading of the SIR complex along the chromatin fiber through the two distinct histone-binding domains in Sir3.


Subject(s)
Chromatin/metabolism , Gene Silencing , Mutation , Nucleosomes/metabolism , Saccharomyces cerevisiae/metabolism , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Chromatin/genetics , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Sequence Alignment , Silent Information Regulator Proteins, Saccharomyces cerevisiae/chemistry
16.
J Proteome Res ; 6(3): 1190-7, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17330950

ABSTRACT

Protein phosphorylation is essential for numerous cellular processes. Large-scale profiling of phosphoproteins continues to enhance the depth and speed at which we understand these processes. The development of effective phosphoprotein and peptide enrichment techniques and improvements to mass spectrometric instrumentation have intensified phosphoproteomic research in recent years, leading to unprecedented achievements. Here, we describe a large-scale phosphorylation analysis of alpha-factor-arrested yeast. Using a multidimensional separation strategy involving preparative SDS-PAGE for prefractionation, in-gel digestion with trypsin, and immobilized metal affinity chromatography (IMAC) enrichment of phosphopeptides, followed by LC-MS/MS analysis employing a hybrid LTQ-Orbitrap mass spectrometer, we were able to catalog a substantial portion of the phosphoproteins present in yeast whole-cell lysate. This analysis yielded the confident identification of 2288 nonredundant phosphorylation sites from 985 proteins. The ambiguity score (Ascore) algorithm was utilized to determine the certainty of site localization for the entire data set. In addition, the size of the data set permitted extraction of known and novel kinase motifs using the Motif-X algorithm. Finally, a large number of members of the pheromone signaling pathway were found as phosphoproteins and are discussed.


Subject(s)
Phosphoproteins/analysis , Proteomics/methods , Saccharomyces cerevisiae Proteins/analysis , Chromatography , Electrophoresis, Polyacrylamide Gel , Pheromones , Phosphopeptides/analysis , Phosphorylation , Signal Transduction , Tandem Mass Spectrometry , Trypsin/metabolism
17.
Mol Cell Biol ; 25(11): 4514-28, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15899856

ABSTRACT

Budding yeast silent chromatin, or heterochromatin, is composed of histones and the Sir2, Sir3, and Sir4 proteins. Their assembly into silent chromatin is believed to require the deacetylation of histones by the NAD-dependent deacetylase Sir2 and the subsequent interaction of Sir3 and Sir4 with these hypoacetylated regions of chromatin. Here we explore the role of interactions among the Sir proteins in the assembly of the SIR complex and the formation of silent chromatin. We show that significant fractions of Sir2, Sir3, and Sir4 are associated together in a stable complex. When the assembly of Sir3 into this complex is disrupted by a specific mutation on the surface of the C-terminal coiled-coil domain of Sir4, Sir3 is no longer recruited to chromatin and silencing is disrupted. Because in sir4 mutant cells the association of Sir3 with chromatin is greatly reduced despite the partial Sir2-dependent deacetylation of histones near silencers, we conclude that histone deacetylation is not sufficient for the full recruitment of silencing proteins to chromatin and that Sir-Sir interactions are essential for the assembly of heterochromatin.


Subject(s)
Gene Silencing , Heterochromatin/metabolism , Histone Deacetylases/metabolism , Saccharomyces cerevisiae/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Sirtuins/metabolism , Acetylation , Histones/metabolism , Mutation , Saccharomyces cerevisiae/metabolism , Silencer Elements, Transcriptional , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Sirtuin 2
18.
Mol Cell Proteomics ; 4(3): 246-54, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15542864

ABSTRACT

Sumoylation represents a vital post-translational modification that pervades numerous aspects of cell biology, including protein targeting, transcriptional regulation, signal transduction, and cell division. However, despite its broad reaching effects, most biological outcomes of protein sumoylation remain poorly understood. In an effort to provide further insight into this complex process, a proteomics approach was undertaken to identify the targets of sumoylation en mass. Specifically, SUMO-conjugated proteins were isolated by a double-affinity purification procedure from a Saccharomyces cerevisiae strain engineered to express tagged SUMO. The components of the isolated protein mixture were then identified by subsequent LC-MS/MS analysis using an LTQ FT mass spectrometer. In this manner, 159 candidate sumoylated proteins were identified by two or more peptides. Furthermore, the high accuracy of the instrument, combined with stringent search criteria, enabled the identification of an additional 92 putative candidates by only one peptide. The validity of this proteomics approach was confirmed by performing subsequent Western blot experiments for numerous proteins and determining the actual sumoylation sites for several other substrates. These data combine with recent works to further our understanding of the breadth and impact of protein sumoylation in a diverse array of biological processes.


Subject(s)
Protein Processing, Post-Translational , Saccharomyces cerevisiae/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Amino Acid Sequence , Chromatography, Liquid , Mass Spectrometry , Molecular Sequence Data , Proteomics
19.
Novartis Found Symp ; 259: 48-56; discussion 56-62, 163-9, 2004.
Article in English | MEDLINE | ID: mdl-15171246

ABSTRACT

Gene silencing involves the assembly of DNA into specialized chromatin domains that are inaccessible to trans-acting factors and are epigenetically inherited. In the budding yeast Saccharomyces cerevisiae, silent heterochromatic DNA domains occur at telomeres, the silent mating type loci, and the rDNA repeats. At telomeres and the mating type loci, silencing requires the Sir2, Sir3 and Sir4 proteins, the conserved N-termini of histones H3 and H4, and a number of chromatin assembly factors. The Sir proteins form a multimeric complex that binds preferentially to deacetylated nucleosomes through the Sir3 and Sir4 subunits. The Sir2 subunit possesses an unusual NAD-dependent deacetylase activity that is required for silencing at each of the above loci. Recent studies have shown that silent chromatin domains are assembled in a step-wise manner involving sequential cycles of deacetylation and SIR complex binding. Sir2-dependent deacetylation is specifically required for the spreading of the complex to regions beyond nucleation sites but not for its initial binding to DNA at the mating type loci and telomeres. A distinct Sir2 complex called RENT is required for silencing at rDNA. In contrast to telomeres and the mating type loci, Sir2 activity is not required for association of RENT with rDNA.


Subject(s)
Chromatin Assembly and Disassembly/physiology , Heterochromatin/metabolism , Saccharomyces cerevisiae/metabolism , Histone Deacetylases/metabolism , Histones/metabolism , Models, Biological , Saccharomyces cerevisiae/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Sirtuin 2 , Sirtuins/metabolism
20.
Mol Cell Biol ; 22(12): 4167-80, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12024030

ABSTRACT

Transcriptional silencing at the budding yeast silent mating type (HM) loci and telomeric DNA regions requires Sir2, a conserved NAD-dependent histone deacetylase, Sir3, Sir4, histones H3 and H4, and several DNA-binding proteins. Silencing at the yeast ribosomal DNA (rDNA) repeats requires a complex containing Sir2, Net1, and Cdc14. Here we show that the native Sir2/Sir4 complex is composed solely of Sir2 and Sir4 and that native Sir3 is not associated with other proteins. We further show that the initial binding of the Sir2/Sir4 complex to DNA sites that nucleate silencing, accompanied by partial Sir2-dependent histone deacetylation, occurs independently of Sir3 and is likely to be the first step in assembly of silent chromatin at the HM loci and telomeres. The enzymatic activity of Sir2 is not required for this initial binding, but is required for the association of silencing proteins with regions distal from nucleation sites. At the rDNA repeats, we show that histone H3 and H4 tails are required for silencing and rDNA-associated H4 is hypoacetylated in a Sir2-dependent manner. However, the binding of Sir2 to rDNA is independent of its histone deacetylase activity. Together, these results support a stepwise model for the assembly of silent chromatin domains in Saccharomyces cerevisiae.


Subject(s)
Chromatin/metabolism , Fungal Proteins/metabolism , Histone Deacetylases/metabolism , Silent Information Regulator Proteins, Saccharomyces cerevisiae , Trans-Activators/metabolism , Yeasts/genetics , Acetylation , Chromatin/genetics , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Gene Expression Regulation, Fungal , Gene Silencing , Histone Deacetylases/genetics , Histones/metabolism , Sirtuin 2 , Sirtuins , Trans-Activators/genetics , Trans-Activators/isolation & purification , Yeasts/metabolism
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