scholarly journals Partial purification and substrate specificity of a ubiquitin hydrolase from Saccharomyces cerevisiae

1991 ◽  
Vol 273 (3) ◽  
pp. 615-620 ◽  
Author(s):  
N Agell ◽  
C Ryan ◽  
M J Schlesinger
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Partial Purification ◽  
Histone H2a ◽  
Polyubiquitin Chain ◽  
Ubiquitinated Protein ◽  
Protein Conjugates ◽  
Isopeptide Bond ◽  
Isopeptide Bonds ◽  
Ubiquitin Molecule ◽  
Intracellular Proteolysis

A ubiquitin hydrolase that removes ubiquitin from a multi-ubiquitinated protein has been purified 600-fold from Saccharomyces cerevisiae. Four different ubiquitin-protein conjugates were assayed as substrates during the purification procedure. Enzymic activities that removed ubiquitin from ubiquitinated histone H2A, a ubiquitin-ubiquitin dimer and a ubiquitin-ribosomal fusion protein were separated during the purification from an activity that removed a single ubiquitin molecule linked by an isopeptide bond to a ubiquitinated protein. The size of the native enzyme was 160 kDa, based on its sedimentation in a sucrose gradient, and the subunit molecular mass was estimated to be 160 kDa, based on a profile of proteins eluted in different fractions by thiol-affinity chromatography. The partially purified hydrolase was not inhibited by a variety of protease inhibitors, except for thiol-blocking reagents. The natural substrate for this enzyme may be the polyubiquitin chain containing ubiquitin molecules bound to each other in isopeptide bonds, with one of them linked to a lysine residue of a protein targeted for intracellular proteolysis.

scholarly journals New de-ubiquitinating enzyme, ubiquitin C-terminal hydrolase 8, in chick skeletal muscle

1997 ◽  
Vol 325 (2) ◽  
pp. 325-330 ◽  
Author(s):  
Sung Hee BAEK ◽  
Seung Kyoon WOO ◽  
Jae LEE ◽  
Yung Joon YOO ◽  
Choong Myung CHO ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Ubiquitinated Protein ◽  
Protein Conjugates ◽  
Isopeptide Bonds ◽  
Linear Poly ◽  
Single Polypeptide ◽  
Free Ubiquitin

We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). Here we report the purification and characterization of one of the UCHs, called UCH-8, with 125I-labelled ubiquitin-α-NH-MHISPPEPESEEEEEHYC as a substrate. The purified UCH-8 behaved as a 240 kDa protein on a Superdex-200 column under non-denaturing conditions but as a 130 kDa polypeptide on analysis by PAGE under denaturing conditions, suggesting that the enzyme consists of two identical subunits. Thus this enzyme seems to be distinct in its dimeric nature from other purified UCHs that consist of a single polypeptide, except that UCH-6 is also a homodimer of 27 kDa subunits. UCH-8 was maximally active between pH 7.5 and 8, but showed little or no activity below pH 7 and above pH 9. Like other UCHs it was sensitive to inhibition by thiol-blocking agents such as N-ethylmaleimide, and by ubiquitin aldehyde. The purified UCH-8 hydrolysed not only ubiquitin-α-NH-protein extensions, including ubiquitin-α-NH-carboxy extension protein of 80 amino acid residues and ubiquitin-α-NH-dihydrofolate reductase, but also branched poly-ubiquitin that are ligated to proteins through ϵ-NH-isopeptide bonds. However, it showed little or no activity against poly-His-tagged di-ubiquitin, suggesting that UCH-8 is not involved in the generation of free ubiquitin from the linear poly-ubiquitin precursors. These results suggest that UCH-8 might have an important role in the production of free ubiquitin and ribosomal proteins from their conjugates as well as in the recycling of ubiquitin molecules after the degradation of poly-ubiquitinated protein conjugates by the 26 S proteasome.

scholarly journals Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase

2021 ◽  
Author(s):  
Zhejian Ji ◽  
Hao Li ◽  
Daniele Peterle ◽  
Joao A Paulo ◽  
Scott B Ficarro ◽  
...  
Keyword(s):  
Polyubiquitin Chain ◽  
Polyubiquitin Chains ◽  
Macromolecular Complexes ◽  
Protein Substrates ◽  
Double Ring ◽  
Isopeptide Bond ◽  
C Terminus ◽  
Ubiquitin Molecule ◽  
Central Pore

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains, rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C-terminus through the central pore of the hexameric double-ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.

scholarly journals A New Saccharomyces cerevisiae Strain with a Mutant Smt3-Deconjugating Ulp1 Protein Is Affected in DNA Replication and Requires Srs2 and Homologous Recombination for Its Viability

2004 ◽  
Vol 24 (12) ◽  
pp. 5130-5143 ◽  
Author(s):  
Christine Soustelle ◽  
Laurence Vernis ◽  
Karine Fréon ◽  
Anne Reynaud-Angelin ◽  
Roland Chanet ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Recombination Mechanism ◽  
Growth Defects ◽  
Protein Conjugates ◽  
Synthetically Lethal ◽  
Mutant Cells ◽  
Insight Into

ABSTRACT The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination. In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion. One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates. This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins. The mutant is unable to grow at 37°C. At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis. Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant. Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant. Upon replication, mutant cells accumulate single-stranded DNA interruptions. These structures are believed to generate different recombination intermediates. Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway.

scholarly journals Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (8) ◽  
pp. 4357-4365 ◽  
Author(s):  
D Huang ◽  
I Farkas ◽  
P J Roach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Mutant Gene ◽  
Glycogen Synthase Kinase ◽  
Partial Purification ◽  
Glycogen Accumulation ◽  
Dependent Protein

In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p.

scholarly journals A new class of histone H2A mutations in Saccharomyces cerevisiae causes specific transcriptional defects in vivo.

1995 ◽  
Vol 15 (4) ◽  
pp. 1999-2009 ◽  
Author(s):  
J N Hirschhorn ◽  
A L Bortvin ◽  
S L Ricupero-Hovasse ◽  
F Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Activation Function ◽  
Histone H2a ◽  
Yeast Saccharomyces Cerevisiae ◽  
New Class ◽  
Suc2 Promoter ◽  
Encoding Gene ◽  
Transcription Activation Function

Nucleosomes have been shown to repress transcription both in vitro and in vivo. However, the mechanisms by which this repression is overcome are only beginning to be understood. Recent evidence suggests that in the yeast Saccharomyces cerevisiae, many transcriptional activators require the SNF/SWI complex to overcome chromatin-mediated repression. We have identified a new class of mutations in the histone H2A-encoding gene HTA1 that causes transcriptional defects at the SNF/SWI-dependent gene SUC2. Some of the mutations are semidominant, and most of the predicted amino acid changes are in or near the N- and C-terminal regions of histone H2A. A deletion that removes the N-terminal tail of histone H2A also caused a decrease in SUC2 transcription. Strains carrying these histone mutations also exhibited defects in activation by LexA-GAL4, a SNF/SWI-dependent activator. However, these H2A mutants are phenotypically distinct from snf/swi mutants. First, not all SNF/SWI-dependent genes showed transcriptional defects in these histone mutants. Second, a suppressor of snf/swi mutations, spt6, did not suppress these histone mutations. Finally, unlike in snf/swi mutants, chromatin structure at the SUC2 promoter in these H2A mutants was in an active conformation. Thus, these H2A mutations seem to interfere with a transcription activation function downstream or independent of the SNF/SWI activity. Therefore, they may identify an additional step that is required to overcome repression by chromatin.

scholarly journals NuA4-Directed Chromatin Transactions throughout the Saccharomyces cerevisiae Genome

2007 ◽  
Vol 27 (15) ◽  
pp. 5327-5335 ◽  
Author(s):  
Melissa Durant ◽  
B. Franklin Pugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Chromatin Remodeling ◽  
Histone Acetyltransferases ◽  
Histone H2a ◽  
Preinitiation Complex ◽  
General Transcription Factor ◽  
Bromodomain Proteins ◽  
Genomic Scale ◽  
H3 Acetylation

ABSTRACT Two of the major histone acetyltransferases in Saccharomyces cerevisiae are NuA4 and SAGA, which acetylate histones H4 and H3, respectively. Acetylated H3 and H4 tails have been implicated in binding bromodomain proteins, including Bdf1. Bdf1 interacts with the general transcription factor TFIID, which might promote preinitiation complex (PIC) assembly. Bdf1 also interacts with the SWR complex (SWR-C). SWR-C is responsible for the deposition of the histone H2A variant H2A.Z. The placement of these interactions into a connected pathway of PIC assembly has not been fully established. Moreover, it is not known how widespread and how variable such a pathway might be on a genomic scale. Here we provide genomic evidence for S. cerevisiae that PIC assembly (TFIID occupancy) and chromatin remodeling (SWR-C and H2A.Z occupancy) are linked in large part to NuA4-directed H4 acetylation and subsequent Bdf1 binding, rather than through SAGA-directed H3 acetylation. Bdf1 and its homolog Bdf2 tend to have distinct locations in the genome. However, the deletion of BDF1 leads to the accumulation of Bdf2 at Bdf1-vacated sites. Thus, while Bdf1 and Bdf2 are at least partially redundant in function, their functions in the genome are geographically distinct.

A role for Saccharomyces cerevisiae histone H2A in DNA repair

Nature ◽  
2000 ◽  
Vol 408 (6815) ◽  
pp. 1001-1004 ◽  
Author(s):  
Jessica A. Downs ◽  
Noel F. Lowndes ◽  
Stephen P. Jackson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Histone H2a

scholarly journals Molecular Strategy for Blocking Isopeptide Bond Formation in Nascent Pilin Proteins

2018 ◽  
Author(s):  
Jaime Andrés Rivas-Pardo ◽  
Carmen L. Badilla ◽  
Rafael Tapia-Rojo ◽  
Álvaro Alonso-Caballero ◽  
Julio M. Fernández
Keyword(s):  
Bacterial Adhesion ◽  
Oxygen Radicals ◽  
Rational Design ◽  
Mechanical Stability ◽  
Host Cells ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Isopeptide Bond ◽  
Isopeptide Bonds ◽  
Single Polypeptide

ABSTRACTBacteria anchor to their host cells through their adhesive pili, which must resist the large mechanical stresses induced by the host as it attempts to dislodge the pathogens. The pili of Gram-positive bacteria are constructed as a single polypeptide made of hundreds of pilin repeats, which contain intramolecular isopeptide bonds strategically located in the structure to prevent their unfolding under force, protecting the pilus from degradation by extant proteases and oxygen radicals. Here, we demonstrate the design of a short peptide that blocks the formation of the isopeptide bond present in the pilin Spy0128 from the human pathogen Streptococcus pyogenes, resulting in mechanically labile pilin domains. We use a combination of protein engineering and AFM force spectroscopy to demonstrate that the peptide blocks the formation of the native isopeptide bond and compromises the mechanics of the domain. While an intact Spy0128 is inextensible at any force, peptide-modified Spy0128 pilins readily unfold at very low forces, marking the abrogation of the intramolecular isopeptide bond as well as the absence of a stable pilin fold. We propose that isopeptide-blocking peptides could be further developed as a novel type of highly-specific anti-adhesive antibiotics to treat Gram-positive pathogens.SignificanceAt the onset of an infection, Gram-positive bacteria adhere to host cells through their pili, filamentous structures built by hundreds of repeats of pilin proteins. These proteins can withstand large mechanical challenges without unfolding, remaining anchored to the host and resisting cleavage by proteases and oxygen radicals present in the targeted tissues. The key structural component that gives pilins mechanical resilience are internal isopeptide bonds, strategically placed so that pilins become inextensible structures. We target this bond by designing a blocking peptide that interferes with its formation during folding. We demonstrate that peptide-modified pilins lack mechanical stability and extend at low forces. We propose this strategy as a rational design of mechanical antibiotics, targeting the Achilles’ Heel of bacterial adhesion.

Sign in / Sign up

Export Citation Format

Share Document