scholarly journals Functional Implications and Ubiquitin-Dependent Degradation of the Peptide Transporter Ptr2 in Saccharomyces cerevisiae

2014 ◽  
Vol 13 (11) ◽  
pp. 1380-1392 ◽  
Author(s):  
Ken Kawai ◽  
Atsuto Moriya ◽  
Satoshi Uemura ◽  
Fumiyoshi Abe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Peptide Binding ◽  
Salt Bridge ◽  
Streptococcus Thermophilus ◽  
Peptide Transporter ◽  
Amino Acid Residues ◽  
Content Type ◽  
Mutant Proteins ◽  
Peptide Binding Site ◽  
In The Wild

ABSTRACTThe peptide transporter Ptr2 plays a central role in di- or tripeptide import inSaccharomyces cerevisiae. AlthoughPTR2transcription has been extensively analyzed in terms of upregulation by the Ubr1-Cup9 circuit, the structural and functional information for this transporter is limited. Here we identified 14 amino acid residues required for peptide import through Ptr2 based on the crystallographic information ofStreptococcus thermophiluspeptide transporter PepTstand based on the conservation of primary sequences among the proton-dependent oligopeptide transporters (POTs). Expression of Ptr2 carrying one of the 14 mutations of which the corresponding residues of PepTstare involved in peptide recognition, salt bridge interaction, or peptide translocation failed to enableptr2Δtrp1cell growth in alanyl-tryptophan (Ala-Trp) medium. We observed that Ptr2 underwent rapid degradation after cycloheximide treatment (half-life, approximately 1 h), and this degradation depended on Rsp5 ubiquitin ligase. The ubiquitination of Ptr2 most likely occurs at the N-terminal lysines 16, 27, and 34. Simultaneous substitution of arginine for the three lysines fully prevented Ptr2 degradation. Ptr2 mutants of the presumed peptide-binding site (E92Q, R93K, K205R, W362L, and E480D) exhibited severe defects in peptide import and were subjected to Rsp5-dependent degradation when cells were moved to Ala-Trp medium, whereas, similar to what occurs in the wild-type Ptr2, mutant proteins of the intracellular gate were upregulated. These results suggest that Ptr2 undergoes quality control and the defects in peptide binding and the concomitant conformational change render Ptr2 subject to efficient ubiquitination and subsequent degradation.

scholarly journals Harpin of Pseudomonas syringae pv. phaseolicola Harbors a Protein Binding Site

2005 ◽  
Vol 18 (1) ◽  
pp. 60-66 ◽  
Author(s):  
Chun-Mei Li ◽  
Minna Haapalainen ◽  
Justin Lee ◽  
Thorsten Nürnberger ◽  
Martin Romantschuk ◽  
...  
Keyword(s):  
Binding Site ◽  
Pseudomonas Syringae ◽  
Peptide Binding ◽  
Phage Display Library ◽  
Mutant Proteins ◽  
Protein Protein Interaction ◽  
Small Proteins ◽  
Peptide Binding Site ◽  
Binding Peptides ◽  
Consensus Amino Acid

Harpin HrpZ of plant-pathogenic bacterium Pseudomonas syringae elicits a hypersensitive response (HR) in some nonhost plants, but its function in the pathogenesis process is still obscure. HrpZ-interacting proteins were identified by screening a phage-display library of random peptides. HrpZ of the bean pathogen P. syringae pv. Phaseolicola (HrpZPph) shows affinity to peptides with a consensus amino acid motif W(L)ARWLL(G/L). To localize the peptide-binding site, the hrpZPph gene was mutagenized with randomly placed 15-bp insertions, and the mutant proteins were screened for the peptide-binding ability. Mutations that inhibited peptide-binding localized to the central region of hrpZPph, which is separate from the previously determined HR-inducing region. Antiserum raised against one of the hrpZPph-binding peptides recognized small proteins in bean, tomato, parsley, and Arabidopsis thaliana but none in tobacco. On native protein blots, hrpZPph bound to a bean protein with similar pI as the protein recognized by the peptide antiserum. The result suggests a protein-protein interaction between the harpin and a host plant protein, possibly involved in the bacterial pathogenesis.

scholarly journals MhbT Is a Specific Transporter for 3-Hydroxybenzoate Uptake by Gram-Negative Bacteria

2012 ◽  
Vol 78 (17) ◽  
pp. 6113-6120 ◽  
Author(s):  
Ying Xu ◽  
Xiaoli Gao ◽  
Song-He Wang ◽  
Hong Liu ◽  
Peter A. Williams ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Hypothetical Protein ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Metabolic Intermediate ◽  
Content Type ◽  
Mutant Proteins ◽  
Green Fluorescent ◽  
Cytoplasmic Loops ◽  
Conserved Residue

ABSTRACTKlebsiella pneumoniaeM5a1 is capable of utilizing 3-hydroxybenzoate via gentisate, and the 6.3-kb gene clustermhbRTDHIMconferred the ability to grow on 3-hydroxybenzoate toEscherichia coliandPseudomonas putidaPaW340. Four of the six genes (mhbDHIM) encode enzymes converting 3-hydroxybenzoate to pyruvate and fumarate via gentisate. MhbR is a gene activator, and MhbT is a hypothetical protein belonging to the transporter of the aromatic acid/H+symporter family. Since a transporter for 3-hydrxybenzoate uptake has not been characterized to date, we investigated whether MhbT is responsible for the uptake of 3-hydroxybenzoate, its metabolic intermediate gentisate, or both. The MhbT-green fluorescent protein (GFP) fusion protein was located on the cytoplasmic membrane.P. putidaPaW340 containingmhbRΔTDHIMcould not grow on 3-hydroxybenzoate; however, supplyingmhbTintransallowed the bacterium to grow on the substrate.K. pneumoniaeM5a1 andP. putidaPaW340 containing recombinant MhbT transported14C-labeled 3-hydroxybenzoate but not14C-labeled gentisate and benzoate into the cells. Site-directed mutagenesis of two conserved amino acid residues (Asp-82 and Asp-314) and a less-conserved residue (Val-311) among the members of the symporter family in the hydrophilic cytoplasmic loops resulted in the loss of 3-hydroxybenzoate uptake byP. putidaPaW340 carrying the mutant proteins. Hence, we demonstrated that MhbT is a specific 3-hydroxybenzoate transporter.

scholarly journals The Lid Subdomain of DnaK Is Required for the Stabilization of the Substrate-binding Site

2004 ◽  
Vol 279 (19) ◽  
pp. 19600-19606 ◽  
Author(s):  
Fernando Moro ◽  
Vanesa Fernández-Sáiz ◽  
Arturo Muga
Keyword(s):  
Binding Site ◽  
Peptide Binding ◽  
Binding Domain ◽  
Peptide Substrate ◽  
Scanning Calorimetry ◽  
Peptide Substrates ◽  
Mutant Proteins ◽  
Functional Implications ◽  
Peptide Binding Site ◽  
Similar Affinity

We examined the effect of deletion of different segments in the helical subdomain (the so-called “lid”) of the DnaK peptide-binding domain on peptide binding and protein stability. At 25 °C, wt DnaK and the deletion mutant proteins are able to stably bind peptides with similar affinity. However, at physiological (37 °C) and stress (42 °C) temperatures, removal of the N-terminal half of αB and the rest of the lid drastically decreases the ability of the protein to bind substrates. Differential scanning calorimetry and infrared spectroscopy show that this behavior is accompanied by destabilization of the peptide-binding domain. Our data suggest that the reversible interaction between the lid and β-sandwich subdomains of DnaK peptide-binding domain is required for the stabilization of the loops that form the peptide-binding site, which in turn modulates the protein affinity for peptide substrates. This interaction might have functional implications because it could prevent rebinding of the peptide substrate, which would be forced to fold.

scholarly journals Enhancement of Acetic Acid Tolerance in Saccharomyces cerevisiae by Overexpression of theHAA1Gene, Encoding a Transcriptional Activator

2012 ◽  
Vol 78 (22) ◽  
pp. 8161-8163 ◽  
Author(s):  
Koichi Tanaka ◽  
Yukari Ishii ◽  
Jun Ogawa ◽  
Jun Shima
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Transcriptional Activator ◽  
Wild Type ◽  
Acetic Acid Tolerance ◽  
Content Type ◽  
In The Wild ◽  
Overexpressing Strain

ABSTRACTHaa1 is a transcriptional activator required forSaccharomyces cerevisiaeadaptation to weak acids. Here we show that the constitutiveHAA1-overexpressing strain acquired a higher level of acetic acid tolerance. Under conditions of acetic acid stress, the intracellular level of acetic acid was significantly lower inHAA1-overexpressing cells than in the wild-type cells.

Rapid Elaboration of Fragments into Leads Applied to Bromodomain-3 Extra Terminal Domain

2020 ◽  
Author(s):  
Luke Adams ◽  
Lorna E. Wilkinson-White ◽  
Menachem J. Gunzburg ◽  
Stephen J. Headey ◽  
Martin J. Scanlon ◽  
...  
Keyword(s):  
Binding Site ◽  
Systematic Approach ◽  
Structural Information ◽  
Peptide Binding ◽  
Chemical Diversity ◽  
Chemical Probes ◽  
Protein Targets ◽  
Structure Activity ◽  
Peptide Binding Site ◽  
Selection Of

The development of low-affinity fragment hits into higher affinity leads is a major hurdle in fragment-based drug design. Here we demonstrate an approach for the Rapid Elaboration of Fragments into Leads (REFiL) applying an integrated workflow that provides a systematic approach to generate higher-affinity binders without the need for structural information. The workflow involves the selection of commercial analogues of fragment hits to generate preliminary structure-activity relationships. This is followed by parallel microscale chemistry using chemoinformatically designed reagent libraries to rapidly explore chemical diversity. Upon completion of a fragment screen against Bromodomain-3 extra terminal (BRD3-ET) domain we applied the REFiL workflow, which allowed us to develop a series of tetrahydrocarbazole ligands that bind to the peptide binding site of BRD3-ET. With REFiL we were able to rapidly improve binding affinity >30-fold. The REFiL workflow can be applied readily to a broad range of protein targets without the need of a structure, allowing the efficient evolution of low-affinity fragments into higher affinity leads and chemical probes.<br>

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

Allotopic antagonism of the non-peptide atrial natriuretic peptide (ANP) antagonist HS-142-1 on natriuretic peptide receptor NPR-A

2002 ◽  
Vol 362 (2) ◽  
pp. 231-237 ◽  
Author(s):  
Hugo POIRIER ◽  
Jean LABRECQUE ◽  
Julie DESCHÊNES ◽  
André DeLÉAN
Keyword(s):  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Mode Of Action ◽  
Transmembrane Domain ◽  
Peptide Binding ◽  
Natriuretic Peptide Receptor ◽  
Receptor Dimerization ◽  
Peptide Receptor ◽  
Peptide Binding Site ◽  
Atrial Natriuretic

The microbial polysaccharide HS-142-1 has been documented as an antagonist of natriuretic peptides. It inhibits activation and peptide binding to both guanylate receptors natriuretic peptide receptor (NPR)-A and NPR-B, but has no effect on the non-cyclase receptor NPR-C. At first sight the effect of HS-142-1 on peptide binding appears to be surmountable, suggesting that it might be competitive despite its chemically divergent nature. We explored its mode of action on wild-type NPR-A (WT), on a disulphide-bridged constitutively active mutant (C423S) and on truncated mutants lacking either their cytoplasmic domain (ΔKC) or both the cytoplasmic and the transmembrane domains (ECD). On the WT, HS-142-1 inhibited atrial natriuretic peptide (ANP) binding with a pK value of 6.51±0.07 (Kd = 0.31μM). It displayed a similar effect on the C423S mutant (pK = 6.31±0.11), indicating that its action might not be due to interference with receptor dimerization. HS-142-1 also inhibited ANP binding to ΔKC with a pK of 7.05±0.05 (Kd = 0.089μM), but it was inactive on ANP binding to ECD at a concentration of 10−4M, suggesting that the antagonism was not competitive at the peptide-binding site located on the ECD and that the transmembrane domain might be required. HS-142-1 also enhanced dissociation of NPR-A-bound 125I-ANP in the presence of excess unlabelled ANP, implying an allotopic (allosteric) mode of action for the antagonist.

scholarly journals Influences of Histone Stoichiometry on the Target Site Preference of Retrotransposons Ty1 and Ty2 in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 761-776 ◽  
Author(s):  
Lori A Rinckel ◽  
David J Garfinkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Promoter Region ◽  
Target Site ◽  
Site Preference ◽  
Wild Type ◽  
Histone Proteins ◽  
Protein Levels ◽  
In The Wild ◽  
Type Strains ◽  
Orientation Bias

Abstract In Saccharomyces cerevisiae, the target site specificity of the retrotransposon Ty1 appears to involve the Ty integration complex recognizing chromatin structures. To determine whether changes in chromatin structure affect Ty1 and Ty2 target site preference, we analyzed Ty transposition at the CAN1 locus in mutants containing altered levels of histone proteins. A Δhta1-htb1 mutant with decreased levels of H2A and H2B histone proteins showed a pattern of Ty1 and Ty2 insertions at CAN1 that was significantly different from that of both the wild-type and a Δhta2-htb2 mutant, which does not have altered histone protein levels. Altered levels of H2A and H2B proteins disrupted a dramatic orientation bias in the CAN1 promoter region. In the wild-type strains, few Ty1 and Ty2 insertions in the promoter region were oriented opposite to the direction of CAN1 transcription. In the Δhta1-htb1 background, however, numerous Ty1 and Ty2 insertions were in the opposite orientation clustered within the TATA region. This altered insertion pattern does not appear to be due to a bias caused by selecting canavanine resistant isolates in the different HTA1-HTB1 backgrounds. Our results suggest that reduced levels of histone proteins alter Ty target site preference and disrupt an asymmetric Ty insertion pattern.

scholarly journals Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily.

1993 ◽  
Vol 13 (4) ◽  
pp. 2554-2563 ◽  
Author(s):  
D Wojciechowicz ◽  
C F Lu ◽  
J Kurjan ◽  
P N Lipke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Adhesion ◽  
Amino Acid ◽  
Cell Surface ◽  
Consensus Sequence ◽  
Binding Domain ◽  
Immunoglobulin Superfamily ◽  
Cell Contact ◽  
Amino Acid Residues ◽  
Adhesion Proteins

alpha-Agglutinin is a cell adhesion glycoprotein expressed on the cell wall of Saccharomyces cerevisiae alpha cells. Binding of alpha-agglutinin to its ligand a-agglutinin, expressed by a cells, mediates cell-cell contact during mating. Analysis of truncations of the 650-amino-acid alpha-agglutinin structural gene AG alpha 1 delineated functional domains of alpha-agglutinin. Removal of the C-terminal hydrophobic sequence allowed efficient secretion of the protein and loss of cell surface attachment. This cell surface anchorage domain was necessary for linkage to a glycosyl phosphatidylinositol anchor. A construct expressing the N-terminal 350 amino acid residues retained full a-agglutinin-binding activity, localizing the binding domain to the N-terminal portion of alpha-agglutinin. A 278-residue N-terminal peptide was inactive; therefore, the binding domain includes residues between 278 and 350. The segment of alpha-agglutinin between amino acid residues 217 and 308 showed significant structural and sequence similarity to a consensus sequence for immunoglobulin superfamily variable-type domains. The similarity of the alpha-agglutinin-binding domain to mammalian cell adhesion proteins suggests that this structure is a highly conserved feature of adhesion proteins in diverse eukaryotes.

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