Biochemical evidence that the whole compartment activity behavior of GAPDH differs between the cytoplasm and nucleus.

Some metabolic enzymes normally occur in the nucleus and cytoplasm. These compartments differ in molecular composition. Since post-translational modification and interaction with allosteric effectors can tune enzyme activity, it follows that the behavior of an enzyme as a catalyst may differ between the cytoplasm and nucleus.

Multienzyme activity profiling for evaluation of cell-to-cell variability of metabolic state.

In solid organs, cells of the same "type" can vary in their molecular phenotype. The basis of this state variation is being revealed by characterizing cell features including the expression pattern of mRNAs and the internal distribution of proteins. Here, the variability of metabolic state between cells is probed by enzyme activity profiling.

Impact of Low-Intensity Pulsed Ultrasound on Transcript and Metabolite Abundance in Saccharomyces cerevisiae.

The interactions of ultrasound with biological materials are exploited for diagnostic, interventional, and therapeutic applications in humans and can improve productivity in industrial-scale generation of organic molecules such as biofuels, vaccines, and antibodies. Accordingly, there is great interest in better understanding the biological effects of ultrasound. We studied the impact of low-intensity pulsed ultrasound (LIPUS) on RNA expression and metabolism of S.

Attenuation of transcriptional and signaling responses limits viability of ρ(0)Saccharomyces cerevisiae during periods of glucose deprivation.

Background: The maintenance of viability during periods when a glycolytic carbon source is limited (or absent) is a major obstacle for cells whose mitochondrial DNA (mtDNA) has been damaged or lost.

Methods: We utilized genome wide transcriptional profiling and in gel mobility analyses to examine the transcriptional response and characterize defects in the phosphorylation dependent signaling events that occur during acute glucose starvation in ρ(0) cells that lack mtDNA. Genetic and pharmacological interventions were employed to clarify the contribution of nutrient responsive kinases to regulation of the transcription factors that displayed abnormal phosphoregulation in ρ(0) cells.

High-Performance Chemical Isotope Labeling Liquid Chromatography-Mass Spectrometry for Profiling the Metabolomic Reprogramming Elicited by Ammonium Limitation in Yeast.

Information about how yeast metabolism is rewired in response to internal and external cues can inform the development of metabolic engineering strategies for food, fuel, and chemical production in this organism. We report a new metabolomics workflow for the characterization of such metabolic rewiring. The workflow combines efficient cell lysis without using chemicals that may interfere with downstream sample analysis and differential chemical isotope labeling liquid chromatography mass spectrometry (CIL LC-MS) for in-depth yeast metabolome profiling.

Rewiring AMPK and mitochondrial retrograde signaling for metabolic control of aging and histone acetylation in respiratory-defective cells.

Abnormal respiratory metabolism plays a role in numerous human disorders. We find that regulation of overall histone acetylation is perturbed in respiratory-incompetent (ρ(0)) yeast. Because histone acetylation is highly sensitive to acetyl-coenzyme A (acetyl-CoA) availability, we sought interventions that suppress this ρ(0) phenotype through reprogramming metabolism.

Promoter regulation by distinct mechanisms of functional interplay between lysine acetylase Rtt109 and histone chaperone Asf1.

The promoter activity of yeast genes can depend on lysine 56 (K56) acetylation of histone H3. This modification of H3 is performed by lysine acetylase Rtt109 acting in concert with histone chaperone Asf1. We have examined the contributions of Rtt109, Asf1, and H3 K56 acetylation to nutrient regulation of a well-studied metabolic gene, ARG1.

SWI/SNF and Asf1 independently promote derepression of the DNA damage response genes under conditions of replication stress.

The histone chaperone Asf1 and the chromatin remodeler SWI/SNF have been separately implicated in derepression of the DNA damage response (DDR) genes in yeast cells treated with genotoxins that cause replication interference. Using genetic and biochemical approaches, we have tested if derepression of the DDR genes in budding yeast involves functional interplay between Asf1 and SWI/SNF. We find that Asf1 and SWI/SNF are both recruited to DDR genes under replication stress triggered by hydroxyurea, and have detected a soluble complex that contains Asf1 and the Snf2 subunit of SWI/SNF.

Genome stability control by checkpoint regulation of tRNA gene transcription.

The RNA polymerase III pre-initiation complex (PIC) assembled on yeast tRNA genes naturally causes replication fork pausing that contributes to genome instability. Mechanistic coupling of the fork pausing activity of tRNA genes to replication has long been considered likely, but only recently demonstrated. In contrast to the expectation that this coupling might occur by a passive mechanism such as direct disruption of transcription factor-DNA complexes by a component of the replisome, it turns out that disassembly of the RNA polymerase III PIC is actively controlled by the replication stress checkpoint signal transduction pathway.

Transcriptional regulation by Asf1: new mechanistic insights from studies of the DNA damage response to replication stress.

Asf1 is a conserved histone H3/H4 chaperone. We find that Asf1 in budding yeast promotes an essential cellular response to replication stress caused by the ribonucleotide reductase inhibitor hydroxyurea. That is, Asf1 stimulates derepression of DNA damage response (DDR) genes during the S phase.

Replication stress checkpoint signaling controls tRNA gene transcription.

In budding yeast, the transcriptional machinery at tRNA genes naturally interferes with replication in a way that can promote chromosome breakage. Here we show that a signaling module composed of core components of the replication stress checkpoint pathway represses this fork-pausing machinery in normally cycling and genotoxin-treated cells. Specifically, the sensor kinase Mec1, the signaling adaptor Mrc1 and the transducer kinase Rad53 relay signals that globally repress tRNA gene transcription during unchallenged proliferation and under conditions of replication stress.

Asf1 can promote trimethylation of H3 K36 by Set2.

Asf1 is a conserved histone H3/H4 chaperone that can assemble and disassemble nucleosomes and promote histone acetylation. Set2 is an H3 K36 methyltransferase. The functions of these proteins intersect in the context of transcription elongation by RNA polymerase II: both contribute to the establishment of repressive chromatin structures that inhibit spurious intragenic transcription.

A glycolytic burst drives glucose induction of global histone acetylation by picNuA4 and SAGA.

Little is known about what enzyme complexes or mechanisms control global lysine acetylation in the amino-terminal tails of the histones. Here, we show that glucose induces overall acetylation of H3 K9, 18, 27 and H4 K5, 8, 12 in quiescent yeast cells mainly by stimulating two KATs, Gcn5 and Esa1. Genetic and pharmacological perturbation of carbon metabolism, combined with (1)H-NMR metabolic profiling, revealed that glucose induction of KAT activity directly depends on increased glucose catabolism.

Untargeted tail acetylation of histones in chromatin: lessons from yeast.

Dynamic acetylation of lysine residues in the amino-terminal tails of the core histones is functionally important for the regulation of diverse DNA-dependent processes in the nucleus, including replication, transcription, and DNA repair. The targeted and untargeted activities of histone lysine acetylases (KATs) and deacetylases (HDACs) both contribute to the dynamics of chromatin acetylation. While the mechanisms and functional consequences of targeted on histone acetylation are well understood, relatively little is known about untargeted histone acetylation.

Chromatin assembly in a crude fraction from yeast cells.

The mechanisms of biological chromatin assembly and their regulation have been studied intensively using cellular extracts, particularly those from the embryonic cells of various metazoans. Here we describe how to prepare and use a crude chromatographic fraction from budding yeast, which also supports biological chromatin assembly. In this system, nucleosomes are assembled by a replication-independent mechanism into physiologically spaced arrays that significantly protect underlying DNA from restriction endonuclease digestion.

Gal4-VP16 directs ATP-independent chromatin reorganization in a yeast chromatin assembly system.

Major insights into the regulation of chromatin organization have stemmed from biochemical studies using Gal4-VP16, a chimeric transcriptional activator in which the DNA binding domain of Gal4p is fused to the activation domain of viral protein VP16. Unexpectedly, given previous intensive efforts to understand how Gal4-VP16 functions in the context of chromatin, we have uncovered a new mode of chromatin reorganization that is dependent on Gal4-VP16. This reorganization is performed by an activity in a crude DEAE (CD) fraction from budding yeast which also supports ATP-dependent assembly of physiologically spaced nucleosome arrays.

Global control of histone modification by the anaphase-promoting complex.

Acetylation and phosphorylation of the amino-terminal tails of the core histones fluctuate on a global scale in concert with other major events in chromosome metabolism. A ubiquitin ligase, the anaphase-promoting complex (APC), controls events in chromosome metabolism such as sister chromatid cohesion and may regulate H3 phosphorylation by targeting Aurora A, one of several S10-directed H3 kinases in vertebrate cells, for destruction by the proteasome. Our analysis of apc10Delta and apc11(ts) loss-of-function mutants reveals that the APC controls the global level of H3 S10 phosphorylation in cycling yeast cells.

Replication-independent assembly of nucleosome arrays in a novel yeast chromatin reconstitution system involves antisilencing factor Asf1p and chromodomain protein Chd1p.

Chromatin assembly in a crude DEAE (CD) fraction from budding yeast is ATP dependent and generates arrays of physiologically spaced nucleosomes which significantly protect constituent DNA from restriction endonuclease digestion. The CD fractions from mutants harboring deletions of the genes encoding histone-binding factors (NAP1, ASF1, and a subunit of CAF-I) and SNF2-like DEAD/H ATPases (SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20) were screened for activity in this replication-independent system. ASF1 deletion substantially inhibits assembly, a finding consistent with published evidence that Asf1p is a chromatin assembly factor.

DNA damage regulation of the RNA components of the translational apparatus: new biology and mechanisms.

It was shown more than 30 years ago that expression of ribosomal (r) RNAs processed from the large precursor rRNA is repressed when eukaryotic cells are exposed to genotoxic stress. More recently it has been found that other RNA components of the translational machinery, the tRNAs and 5S rRNA transcribed by RNA polymerase (pol) III, are also downregulated in cells that have experienced DNA damage. In other words, the DNA damage response involves coordinate repression of genes whose products comprise the heart of the translational machinery.