scholarly journals Proline Reverses the Abnormal Phenotypes of Colletotrichum trifolii Associated with Expression of Endogenous Constitutively Active Ras

2002 ◽  
Vol 68 (4) ◽  
pp. 1647-1651 ◽  
Author(s):  
Stephen D. Memmott ◽  
Young-sil Ha ◽  
Martin B. Dickman
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Signaling Pathways ◽  
G Protein ◽  
Growth Medium ◽  
Causative Organism ◽  
Wild Type ◽  
Colletotrichum Trifolii ◽  
Rich Media ◽  
Constitutively Active

ABSTRACT Colletotrichum trifolii is the causative organism of alfalfa anthracnose. We previously cloned and characterized the small prototypical G protein, Ras, of C. trifolii, which is involved in the signaling pathways that mediate interaction between the pathogen and its host. Transformants expressing constitutively active forms of Ras have growth medium-dependent phenotypes. In nutrient-rich media (e.g., yeast extract and peptone), the phenotype of the transformants was indistinguishable from that of the wild type. However, during nutrient starvation, the transformants lose polarity, have distended hyphae, and fail to sporulate and produce appressoria. Since peptone caused the phenotype to revert, amino acids were tested singly and in combination to identify the responsible amino acid(s). We found that 1.6 mM proline in the medium reverses the constitutively active Ras phenotype.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

scholarly journals Mice with Deficiency of G Protein γ3 Are Lean and Have Seizures

2004 ◽  
Vol 24 (17) ◽  
pp. 7758-7768 ◽  
Author(s):  
William F. Schwindinger ◽  
Kathryn E. Giger ◽  
Kelly S. Betz ◽  
Anna M. Stauffer ◽  
Elaine M. Sunderlin ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Gene Targeting ◽  
Wild Type ◽  
Phenotypic Changes ◽  
Reduced Body ◽  
Γ Subunit ◽  
Body Weights ◽  
The Brain ◽  
Increased Susceptibility

ABSTRACT Emerging evidence suggests that the γ subunit composition of an individual G protein contributes to the specificity of the hundreds of known receptor signaling pathways. Among the twelve γ subtypes, γ3 is abundantly and widely expressed in the brain. To identify specific functions and associations for γ3, a gene-targeting approach was used to produce mice lacking the Gng3 gene (Gng3 −/−). Confirming the efficacy and specificity of gene targeting, Gng3 −/− mice show no detectable expression of the Gng3 gene, but expression of the divergently transcribed Bscl2 gene is not affected. Suggesting unique roles for γ3 in the brain, Gng3 −/− mice display increased susceptibility to seizures, reduced body weights, and decreased adiposity compared to their wild-type littermates. Predicting possible associations for γ3, these phenotypic changes are associated with significant reductions in β2 and αi3 subunit levels in certain regions of the brain. The finding that the Gng3 −/− mice and the previously reported Gng7 −/− mice display distinct phenotypes and different αβγ subunit associations supports the notion that even closely related γ subtypes, such as γ3 and γ7, perform unique functions in the context of the organism.

Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate

2001 ◽  
Vol 281 (4) ◽  
pp. G1034-G1043 ◽  
Author(s):  
Kousei Ito ◽  
Hiroshi Suzuki ◽  
Yuichi Sugiyama
Keyword(s):  
Amino Acids ◽  
Multidrug Resistance ◽  
Amino Acid ◽  
Membrane Vesicles ◽  
Single Amino Acid ◽  
Sf9 Cells ◽  
Transport Activity ◽  
Wild Type ◽  
Cationic Amino Acids ◽  
The Difference

Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.

scholarly journals Amino acids whose intracellular levels change most during aging alter chronological lifespan of fission yeast

2020 ◽  
Author(s):  
Charalampos Rallis ◽  
Michael Mülleder ◽  
Graeme Smith ◽  
Yan Zi Au ◽  
Markus Ralser ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Fission Yeast ◽  
Yeast Cells ◽  
Total Amino Acid ◽  
Wild Type ◽  
Chronological Lifespan ◽  
Biomarkers Of Aging ◽  
Concentration Changes ◽  
Amino Acid Levels

AbstractAmino acid deprivation or supplementation can affect cellular and organismal lifespan, but we know little about the role of concentration changes in free, intracellular amino acids during aging. Here, we determine free amino-acid levels during chronological aging of non-dividing fission yeast cells. We compare wild-type with long-lived mutant cells that lack the Pka1 protein of the protein kinase A signalling pathway. In wild-type cells, total amino-acid levels decrease during aging, but much less so in pka1 mutants. Two amino acids strongly change as a function of age: glutamine decreases, especially in wild-type cells, while aspartate increases, especially in pka1 mutants. Supplementation of glutamine is sufficient to extend the chronological lifespan of wild-type but not of pka1Δ cells. Supplementation of aspartate, on the other hand, shortens the lifespan of pka1Δ but not of wild-type cells. Our results raise the possibility that certain amino acids are biomarkers of aging, and their concentrations during aging can promote or limit cellular lifespan.

scholarly journals Mutational Analysis of the Hydrophobic Tail of the Human Immunodeficiency Virus Type 1 p6Gag Protein Produces a Mutant That Fails To Package Its Envelope Protein

1999 ◽  
Vol 73 (1) ◽  
pp. 19-28 ◽  
Author(s):  
David E. Ott ◽  
Elena N. Chertova ◽  
Laura K. Busch ◽  
Lori V. Coren ◽  
Tracy D. Gagliardi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Human Immunodeficiency Virus Type ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hydrophobic Tail ◽  
Carboxyl Terminal ◽  
Hiv 1

ABSTRACT The p6Gag protein of human immunodeficiency virus type 1 (HIV-1) is produced as the carboxyl-terminal sequence within the Gag polyprotein. The amino acid composition of this protein is high in hydrophilic and polar residues except for a patch of relatively hydrophobic amino acids found in the carboxyl-terminal 16 amino acids. Internal cleavage of p6Gag between Y36 and P37, apparently by the HIV-1 protease, removes this hydrophobic tail region from approximately 30% of the mature p6Gag proteins in HIV-1MN. To investigate the importance of this cleavage and the hydrophobic nature of this portion of p6Gag, site-directed mutations were made at the minor protease cleavage site and within the hydrophobic tail. The results showed that all of the single-amino-acid-replacement mutants exhibited either reduced or undetectable cleavage at the site yet almost all were nearly as infectious as wild-type virus, demonstrating that processing at this site is not important for viral replication. However, one exception, Y36F, was 300-fold as infectious the wild type. In contrast to the single-substitution mutants, a virus with two substitutions in this region of p6Gag, Y36S-L41P, could not infect susceptible cells. Protein analysis showed that while the processing of the Gag precursor was normal, the double mutant did not incorporate Env into virus particles. This mutant could be complemented with surface glycoproteins from vesicular stomatitis virus and murine leukemia virus, showing that the inability to incorporate Env was the lethal defect for the Y36S-L41P virus. However, this mutant was not rescued by an HIV-1 Env with a truncated gp41TM cytoplasmic domain, showing that it is phenotypically different from the previously described MA mutants that do not incorporate their full-length Env proteins. Cotransfection experiments with Y36S-L41P and wild-type proviral DNAs revealed that the mutant Gag dominantly blocked the incorporation of Env by wild-type Gag. These results show that the Y36S-L41P p6Gag mutation dramatically blocks the incorporation of HIV-1 Env, presumably acting late in assembly and early during budding.

scholarly journals Chemical composition and nutritional quality of short-ripened rennet cheeses produced by traditional methods

2018 ◽  
Vol 74 (10) ◽  
pp. 5971-2018 ◽  
Author(s):  
PRZEMYSŁAW KNYSZ ◽  
MICHAŁ GONDEK ◽  
RENATA PYZ-ŁUKASIK ◽  
MONIKA ZIOMEK ◽  
ŁUKASZ DROZD ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Chemical Composition ◽  
G Protein ◽  
Nutritive Value ◽  
Salt Content ◽  
Amino Acid Content ◽  
Traditional Methods ◽  
Dairy Starter Cultures ◽  
Limiting Amino Acids

The aim of the study was to determine the variability in the chemical composition and nutritive value parameters of smoked and unsmoked short-ripened rennet cheeses and unsmoked long-ripened rennet cheeses produced by traditional methods. The raw material for the production of short-ripened cheeses was pasteurized cows’ milk obtained from a dairy, whereas the long-ripened cheeses were manufactured from raw cows’ milk obtained from the producer’s farm. All three varieties of cheese examined were produced with commercial dairy starter cultures. The material for the study was collected in winter, directly at the producers’ retail outlets in southern and eastern Poland. The basic chemical composition was determined according to the Polish Standards, whereas the amino acid profiles of proteins from the cheeses were determined by ion-exchange chromatography. The result analysis revealed significant differences between the different varieties of cheese in terms of their water content, ranging from 32.2% to 42.1%, as well as protein content, which varied from 25.6% to 31.6%. Fat levels ranged between 22.2% and 24%, whereas total ash content amounted to 5.1-5.8%. The significantly highest salt content was found in unsmoked short-ripened cheeses. In all three cheese varieties, the total exogenous amino acid content was comparable, ranging from 46.17 g to 47.36 g/100 g protein, and that of endogenous amino acids varied from 52 g to 53 g/100 g protein. The biological value of proteins was determined by calculating to the chemical score (CS), as described by Mitchell and Block, and the essential amino acid index (EAAI), as described by Oser. A comparison of the results with the standard chicken egg white proteins showed that the limiting amino acids for all varieties of cheese were methionine and cysteine. On the other hand, a comparison with the FAO/WHO-suggested pattern of amino acid requirements (1991) for all age groups over 1 year of age showed that the limiting amino acids were methionine and cysteine in smoked and unsmoked short-ripened cheeses, and treonine in long-ripened cheeses. Considering the chemical indices, such as CS and EAAI, it may be concluded that the traditional rennet cheeses produced in southern and eastern Poland have a favourable amino acid composition of proteins and a high nutritive value..

scholarly journals Two site-directed mutations abrogate enzyme activity but have different effects on the conformation and cellular content of the N-acetylgalactosamine 4-sulphatase protein

1995 ◽  
Vol 307 (2) ◽  
pp. 457-463 ◽  
Author(s):  
D A Brooks ◽  
D A Robertson ◽  
C Bindloss ◽  
T Litjens ◽  
D S Anson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Cell Line ◽  
Protein Level ◽  
Structural Modification ◽  
Wild Type ◽  
Cellular Content ◽  
Control Protein ◽  
Wild Type Control

The sulphatase family of enzymes have regions of sequence similarity, but relatively little is known about either the structure-function relationships of sulphatases, or the role of highly conserved amino acids. The sequence of amino acids CTPSR at position 91-95 of 4-sulphatase has been shown to be highly conserved in all of the sequenced sulphatase enzymes. The cysteine at amino acid 91 of 4-sulphatase was selected for mutation analysis due to its potential role in either the active site, substrate-binding site or part of a key structural domain of 4-sulphatase and due to the absence of naturally occurring mutations in this residue in mucopolysaccharidosis type VI (MPS VI) patients. Two mutations, C91S and C91T, altering amino acid 91 of 4-sulphatase were generated and expressed in Chinese hamster ovary cells. Biochemical analysis of protein from a C91S cell line demonstrated no detectable 4-sulphatase enzyme activity but a relatively normal level of 4-sulphatase polypeptide (180% of the wild-type control protein level). Epitope detection, using a panel of ten monoclonal antibodies, demonstrated that the C91S polypeptide had a similar immunoreactivity to wild-type 4-sulphatase, suggesting that the C91S substitution does not induce a major structural change in the protein. Reduced catalytic activity associated with normal levels of 4-sulphatase protein have not been observed in any of the MPS VI patients tested and all show evidence of structural modification of 4-sulphatase protein with the same panel of antibodies [Brooks, McCourt, Gibson, Ashton, Shutter and Hopwood (1991) Am. J. Hum. Genet. 48, 710-719]. The loss of enzyme activity without a detectable protein conformation change suggests that Cys-91 may be a critical residue in the catalytic process. In contrast, analysis of protein from a C91T cell line revealed low levels of catalytically inactive 4-sulphatase polypeptide (0.37% of the wild-type control protein level) which had missing or masked epitopes, suggesting an altered protein structure or conformation. Subcellular fractionation studies of the C91T cell line demonstrated a high proportion of 4-sulphatase polypeptide content in organelles characteristic of microsomes. The aberrant intracellular localization and the reduced cellular content of 4-sulphatase polypeptide was consistent with the observed structural modification leading to retention and degradation of the protein within an early vacuolar compartment.

scholarly journals Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

1997 ◽  
Vol 17 (1) ◽  
pp. 115-122 ◽  
Author(s):  
M B Sainz ◽  
S A Goff ◽  
V L Chandler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Transcriptional Activation Domain ◽  
Acidic Amino Acids

C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains.

scholarly journals Characterization of human torsinA and its dystonia-associated mutant form

2003 ◽  
Vol 374 (1) ◽  
pp. 117-122 ◽  
Author(s):  
Zhonghua LIU ◽  
Anna ZOLKIEWSKA ◽  
Michal ZOLKIEWSKI
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Signal Sequence ◽  
Eukaryotic Cells ◽  
Membrane Association ◽  
S2 Cells ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Terminal Amino ◽  
Membrane Anchoring

Deletion of a single glutamate in torsinA correlates with early-onset dystonia, the most severe form of a neurological disorder characterized by uncontrollable muscle contractions. TorsinA is targeted to the ER (endoplasmic reticulum) in eukaryotic cells. We investigated the processing and membrane association of torsinA and the dystonia-associated Glu-deletion mutant (torsinAΔE). We found that the signal sequence of torsinA (residues 1–20 from the 40 amino-acid long N-terminal hydrophobic region) is cleaved in Drosophila S2 cells, as shown by the N-terminal sequencing after partial protein purification. TorsinA is not secreted from S2 cells. Consistently, sodium carbonate extraction and Triton X-114 treatment showed that torsinA is associated with the ER membrane in CHO (Chinese-hamster ovary) cells. In contrast, a variant of torsinA that contains the native signal sequence without the hydrophobic region Ile24–Pro40 does not associate with the membranes in CHO cells, and a truncated torsinA without the 40 N-terminal amino acids is secreted in the S2 culture. Thus the 20-amino-acid-long hydrophobic segment in torsinA, which remains at the N-terminus after signal-peptide cleavage, is responsible for the membrane anchoring of torsinA. TorsinAΔE showed similar cleavage of the 20 N-terminal amino acids and membrane association properties similar to wild-type torsinA but, unlike the wild-type, torsinAΔE was not secreted in the S2 culture even after deletion of the membrane-anchoring segment. This indicates that the dystonia-associated mutation produces a structurally distinct, possibly misfolded, form of torsinA, which cannot be properly processed in the secretory pathway of eukaryotic cells.

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