scholarly journals Specificity of Class II Hsp40 Sis1 in Maintenance of Yeast Prion [RNQ +]

2003 ◽  
Vol 14 (3) ◽  
pp. 1172-1181 ◽  
Author(s):  
Nelson Lopez ◽  
Rebecca Aron ◽  
Elizabeth A. Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Molecular Chaperones ◽  
Mammalian Cells ◽  
Single Amino Acid ◽  
Yeast Prion ◽  
Rich Region ◽  
F Region ◽  
Methionine Residues ◽  
Hsp 40

Sis1 and Ydj1, functionally distinct heat shock protein (Hsp)40 molecular chaperones of the yeast cytosol, are homologs of Hdj1 and Hdj2 of mammalian cells, respectively. Sis1 is necessary for propagation of the Saccharomyces cerevisiae prion [RNQ + ]; Ydj1 is not. The ability to function in [RNQ + ] maintenance has been conserved, because Hdj1 can function to maintain Rnq1 in an aggregated form in place of Sis1, but Hdj2 cannot. An extended glycine-rich region of Sis1, composed of a region rich in phenylalanine residues (G/F) and another rich in methionine residues (G/M), is critical for prion maintenance. Single amino acid alterations in a short stretch of amino acids of the G/F region of Sis1 that are absent in the otherwise highly conserved G/F region of Ydj1 cause defects in prion maintenance. However, there is some functional redundancy within the glycine-rich regions of Sis1, because a deletion of the adjacent glycine/methionine (G/M) region was somewhat defective in propagation of [RNQ + ] as well. These results are consistent with a model in which the glycine-rich regions of Hsp40s contain specific determinants of function manifested through interaction with Hsp70s.

scholarly journals The Glycine-Phenylalanine-Rich Region Determines the Specificity of the Yeast Hsp40 Sis1

1999 ◽  
Vol 19 (11) ◽  
pp. 7751-7758 ◽  
Author(s):  
Wei Yan ◽  
Elizabeth A. Craig
Keyword(s):  
Amino Acids ◽  
Molecular Chaperones ◽  
Rich Region ◽  
Critical Determinant ◽  
Unique Region ◽  
Chimeric Genes ◽  
F Region ◽  
Initiation Of Translation ◽  
J Domain ◽  
Essential Function

ABSTRACT Hsp40s are ubiquitous, conserved proteins which function with molecular chaperones of the Hsp70 class. Sis1 is an essential Hsp40 of the cytosol of Saccharomyces cerevisiae, thought to be required for initiation of translation. We carried out a genetic analysis to determine the regions of Sis1 required to perform its key function(s). A C-terminal truncation of Sis1, removing 231 amino acids but retaining the N-terminal 121 amino acids encompassing the J domain and the glycine-phenylalanine-rich (G-F) region, was able to rescue the inviability of a Δsis1 strain. The yeast cytosol contains other Hsp40s, including Ydj1. To determine which regions carried the critical determinants of Sis1 function, we constructed chimeric genes containing portions of SIS1 and YDJ1. A chimera containing the J domain of Sis1 and the G-F region of Ydj1 could not rescue the lethality of the Δsis1 strain. However, a chimera with the J domain of Ydj1 and the G/F region of Sis1 could rescue the strain’s lethality, indicating that the G-F region is a unique region required for the essential function of Sis1. However, a J domain is also required, as mutants expected to cause a disruption of the interaction of the J domain with Hsp70 are inviable. We conclude that the G-F region, previously thought only to be a linker or spacer region between the J domain and C-terminal regions of Hsp40s, is a critical determinant of Sis1 function.

scholarly journals Finding the Binding Site of Peloruside A and its Secondary Effects in Saccharomyces Cerevisiae using a  Chemical Genetics Approach

2021 ◽  
Author(s):  
◽  
Reem Hanna
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Amino Acids ◽  
Flow Cytometry ◽  
Binding Site ◽  
Mammalian Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peloruside A ◽  
Microtubule Function

<p>Peloruside A, a natural product isolated from the marine sponge Mycale hentscheli, is a microtubule-stabilising agent that has a similar mechanism of action to the anticancer drug paclitaxel and is cytotoxic to cultured mammalian cells. Peloruside appears to bind to a distinct site on mammalian tubulin that is different from that of the taxoid-site drugs. Because of the high sequence homology between yeast and mammalian tubulin, Saccharomyces cerevisiae (S. cerevisiae) was used as a model organism to characterise the peloruside-binding site with the aim of advancing our understanding about this site on mammalian tubulin. Wild type S. cerevisiae (BY4741) was sensitive to peloruside at uM concentrations; however, a strain that lacks the mad2 (Mitotic Arrest Deficient 2) gene showed increased sensitivity to the drug at much lower uM concentrations. This gene is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. The main aims of this project were to define the binding site of peloruside A using yeast tubulin to see if microtubule function and/or morphology is altered in yeast by peloruside, and to identify any secondary drug targets "friends of the target" through chemical genetic interactions profiling (Homozygous deletion profiling microarray). Site-directed mutagenesis was used to mutate two conserved amino acids (A296T; R306H) known to confer resistance to peloruside in mammalian cells. Based on a published computer model of the peloruside binding site on mammalian tubulin, we also mutated three other amino acids, two that were predicted to affect peloruside binding (Q291M and N337L), and one that was predicted to affect laulimalide binding but have little affect on peloruside binding (V333W). We also included a negative control that was predicted to have no effect on peloruside binding (R282Q) and would affect epothilone binding. We found that of the six point mutations, only Q291M failed to confer resistance in yeast and instead it increased the inhibition to the drug. Using a bud index assay, confocal microscopy, and flow cytometry, 40-50 uM peloruside was shown to block cells in G2/M of the cell cycle, confirming a direct action of the drug on microtubule function. Homozygous profiling (HOP) microarray analysis of a deletion mutant set of yeast genes was also carried out to identify gene products that interact with peloruside in order to link the drug to specific networks or biochemical pathways in the cells. From site-directed mutagenesis, we concluded that peloruside binds to yeast B-tubulin in the region predicted by the published model of the binding site, and therefore mapping the site on yeast tubulin could provide useful information about the mammalian binding site for peloruside. The bud index, flow cytometry, and confocal microscopy experiments provided further evidence that peloruside interacts with yeast tubulin. From HOP we found that peloruside has roles in the cell cycle, as expected, and has effects on protein transport, secretion, cell wall synthesis, and steroid biosynthesis pathways.</p>

scholarly journals Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae

2012 ◽  
Vol 79 (5) ◽  
pp. 1500-1507 ◽  
Author(s):  
Suk-Jin Ha ◽  
Heejin Kim ◽  
Yuping Lin ◽  
Myoung-Uoon Jang ◽  
Jonathan M. Galazka ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Single Amino Acid ◽  
Kinetic Properties ◽  
Wild Type ◽  
Scheffersomyces Stipitis ◽  
Content Type ◽  
Charged Amino Acids ◽  
Cellodextrin Transporter ◽  
Engineered Strain

ABSTRACTSaccharomyces cerevisiaecannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) genes fromNeurospora crassa. Here, we report that an engineeredS. cerevisiaestrain expressing the putative hexose transporter geneHXT2.4fromScheffersomyces stipitisandgh1-1can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter whenHXT2.4is overexpressed inS. cerevisiae. However, cellobiose fermentation by the engineered strain expressingHXT2.4andgh1-1was much slower and less efficient than that by an engineered strain that initially expressedcdt-1andgh1-1. The rate of cellobiose fermentation by theHXT2.4-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolvedS. cerevisiaestrain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higherKmand 4-fold higherVmaxvalues than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed inS. cerevisiaeare suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineeredS. cerevisiaestrains.

scholarly journals Impact of Amyloid Polymorphism on Prion-Chaperone Interactions in Yeast

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 349 ◽  
Author(s):  
Andrea N. Killian ◽  
Sarah C. Miller ◽  
Justin K. Hines
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Chaperones ◽  
Chaperone Proteins ◽  
Yeast Prion ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genetic Elements ◽  
Yeast Prions ◽  
Amyloid Aggregates ◽  
Structural Differences ◽  
Chaperone Interactions

Yeast prions are protein-based genetic elements found in the baker’s yeast Saccharomyces cerevisiae, most of which are amyloid aggregates that propagate by fragmentation and spreading of small, self-templating pieces called propagons. Fragmentation is carried out by molecular chaperones, specifically Hsp104, Hsp70, and Hsp40. Like other amyloid-forming proteins, amyloid-based yeast prions exhibit structural polymorphisms, termed “strains” in mammalian systems and “variants” in yeast, which demonstrate diverse phenotypes and chaperone requirements for propagation. Here, the known differential interactions between chaperone proteins and yeast prion variants are reviewed, specifically those of the yeast prions [PSI+], [RNQ+]/[PIN+], and [URE3]. For these prions, differences in variant-chaperone interactions (where known) with Hsp104, Hsp70s, Hsp40s, Sse1, and Hsp90 are summarized, as well as some interactions with chaperones of other species expressed in yeast. As amyloid structural differences greatly impact chaperone interactions, understanding and accounting for these variations may be crucial to the study of chaperones and both prion and non-prion amyloids.

scholarly journals Context affects nuclear protein localization in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (2) ◽  
pp. 384-389 ◽  
Author(s):  
M Nelson ◽  
P Silver
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Nuclear Localization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Single Amino Acid ◽  
Nuclear Localization Sequence ◽  
Localization Sequence ◽  
Beta Galactosidase

Proteins destined for the nucleus contain nuclear localization sequences, short stretches of amino acids responsible for targeting them to the nucleus. We show that the first 29 amino acids of GAL4, a yeast DNA-binding protein, function efficiently as a nuclear localization sequence when fused to normally cytoplasmic invertase, but not when fused to Escherichia coli beta-galactosidase. Moreover, the nuclear localization sequence from simian virus 40 T antigen functions better when fused to invertase than when fused to beta-galactosidase. A single amino acid change in the T-antigen nuclear localization sequence inhibits the nuclear localization of simian virus 40-invertase and simian virus 40-beta-galactosidase in Saccharomyces cerevisiae. From these results, we conclude that the relative ability of a nuclear localization sequence to act depends on the protein to which it is linked.

Context affects nuclear protein localization in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (2) ◽  
pp. 384-389
Author(s):  
M Nelson ◽  
P Silver
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Nuclear Localization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Single Amino Acid ◽  
Nuclear Localization Sequence ◽  
Localization Sequence ◽  
Beta Galactosidase

Proteins destined for the nucleus contain nuclear localization sequences, short stretches of amino acids responsible for targeting them to the nucleus. We show that the first 29 amino acids of GAL4, a yeast DNA-binding protein, function efficiently as a nuclear localization sequence when fused to normally cytoplasmic invertase, but not when fused to Escherichia coli beta-galactosidase. Moreover, the nuclear localization sequence from simian virus 40 T antigen functions better when fused to invertase than when fused to beta-galactosidase. A single amino acid change in the T-antigen nuclear localization sequence inhibits the nuclear localization of simian virus 40-invertase and simian virus 40-beta-galactosidase in Saccharomyces cerevisiae. From these results, we conclude that the relative ability of a nuclear localization sequence to act depends on the protein to which it is linked.

Expression of human melanocortin 4 receptor in Saccharomyces cerevisiae

2011 ◽  
Vol 6 (2) ◽  
pp. 167-175 ◽  
Author(s):  
Vita Ignatovica ◽  
Ramona Petrovska ◽  
Davids Fridmanis ◽  
Janis Klovins
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Mammalian Cells ◽  
Energy Homeostasis ◽  
Expression System ◽  
Yeast Saccharomyces Cerevisiae ◽  
Yeast Expression System ◽  
Melanocortin 4 Receptor

AbstractThe melanocortin 4 receptor (MC4R) is involved in the regulation of energy homeostasis and is known as one of the major hypothalamic regulators of food intake. Several studies have shown that replacement of aspartic acid at position 126 of the MC4R abolishes the ligand binding. We used the modified yeast Saccharomyces cerevisiae strain MMY28 to functionally express the MC4R and characterise the importance of this amino acid for ligand based activation of the receptor. The efficiency of the functional expression system was estimated by activation with αMSH, ACTH and THIQ and compared with cAMP response in mammalian cells. We generated the library of MC4R mutants randomised at the amino acid position 126. Recombinant MC4R clones were screened for the αMSH induced activity in yeast. From 9 different amino acids obtained only the natural aspartic acid displayed the ligand dependent activity of MC4R. The MC4R variants with glutamic acid and leucine at position 126, however, displayed higher background activity than other amino acid substitutions. The results suggest that the yeast expression system is suitable for screening of the MC4R receptor ligands and that the substitution of aspartic acid at position 126 of MC4R by different amino acids functionally inactivates the receptor.

The yeast prion protein Ure2: insights into the mechanism of amyloid formation

2011 ◽  
Vol 39 (5) ◽  
pp. 1359-1364 ◽  
Author(s):  
Li-Jun Chen ◽  
Elizabeth B. Sawyer ◽  
Sarah Perrett
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Conformational Change ◽  
Nitrogen Metabolism ◽  
Prion Protein ◽  
Molecular Chaperones ◽  
Fibril Formation ◽  
Regulatory Function ◽  
Amyloid Formation ◽  
Yeast Prion

Ure2, a regulator of nitrogen metabolism, is the protein determinant of the [URE3] prion state in Saccharomyces cerevisiae. Upon conversion into the prion form, Ure2 undergoes a heritable conformational change to an amyloid-like aggregated state and loses its regulatory function. A number of molecular chaperones have been found to affect the prion properties of Ure2. The studies carried out in our laboratory have been aimed at elucidating the structure of Ure2 fibrils, the mechanism of amyloid formation and the effect of chaperones on the fibril formation of Ure2.

scholarly journals Finding the Binding Site of Peloruside A and its Secondary Effects in Saccharomyces Cerevisiae using a  Chemical Genetics Approach

2021 ◽  
Author(s):  
◽  
Reem Hanna
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Amino Acids ◽  
Flow Cytometry ◽  
Binding Site ◽  
Mammalian Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peloruside A ◽  
Microtubule Function

<p>Peloruside A, a natural product isolated from the marine sponge Mycale hentscheli, is a microtubule-stabilising agent that has a similar mechanism of action to the anticancer drug paclitaxel and is cytotoxic to cultured mammalian cells. Peloruside appears to bind to a distinct site on mammalian tubulin that is different from that of the taxoid-site drugs. Because of the high sequence homology between yeast and mammalian tubulin, Saccharomyces cerevisiae (S. cerevisiae) was used as a model organism to characterise the peloruside-binding site with the aim of advancing our understanding about this site on mammalian tubulin. Wild type S. cerevisiae (BY4741) was sensitive to peloruside at uM concentrations; however, a strain that lacks the mad2 (Mitotic Arrest Deficient 2) gene showed increased sensitivity to the drug at much lower uM concentrations. This gene is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. The main aims of this project were to define the binding site of peloruside A using yeast tubulin to see if microtubule function and/or morphology is altered in yeast by peloruside, and to identify any secondary drug targets "friends of the target" through chemical genetic interactions profiling (Homozygous deletion profiling microarray). Site-directed mutagenesis was used to mutate two conserved amino acids (A296T; R306H) known to confer resistance to peloruside in mammalian cells. Based on a published computer model of the peloruside binding site on mammalian tubulin, we also mutated three other amino acids, two that were predicted to affect peloruside binding (Q291M and N337L), and one that was predicted to affect laulimalide binding but have little affect on peloruside binding (V333W). We also included a negative control that was predicted to have no effect on peloruside binding (R282Q) and would affect epothilone binding. We found that of the six point mutations, only Q291M failed to confer resistance in yeast and instead it increased the inhibition to the drug. Using a bud index assay, confocal microscopy, and flow cytometry, 40-50 uM peloruside was shown to block cells in G2/M of the cell cycle, confirming a direct action of the drug on microtubule function. Homozygous profiling (HOP) microarray analysis of a deletion mutant set of yeast genes was also carried out to identify gene products that interact with peloruside in order to link the drug to specific networks or biochemical pathways in the cells. From site-directed mutagenesis, we concluded that peloruside binds to yeast B-tubulin in the region predicted by the published model of the binding site, and therefore mapping the site on yeast tubulin could provide useful information about the mammalian binding site for peloruside. The bud index, flow cytometry, and confocal microscopy experiments provided further evidence that peloruside interacts with yeast tubulin. From HOP we found that peloruside has roles in the cell cycle, as expected, and has effects on protein transport, secretion, cell wall synthesis, and steroid biosynthesis pathways.</p>

scholarly journals Mutational Analysis of the N-Terminal DNA-Binding Domain of Sleeping Beauty Transposase: Critical Residues for DNA Binding and Hyperactivity in Mammalian Cells

2004 ◽  
Vol 24 (20) ◽  
pp. 9239-9247 ◽  
Author(s):  
Stephen R. Yant ◽  
Julie Park ◽  
Yong Huang ◽  
Jacob Giehm Mikkelsen ◽  
Mark A. Kay
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Nuclear Translocation ◽  
Mutational Analysis ◽  
Sleeping Beauty ◽  
Binding Domain ◽  
Single Amino Acid ◽  
Dna Binding Domain

ABSTRACT The N-terminal domain of the Sleeping Beauty (SB) transposase mediates transposon DNA binding, subunit multimerization, and nuclear translocation in vertebrate cells. For this report, we studied the relative contributions of 95 different residues within this multifunctional domain by large-scale mutational analysis. We found that each of four amino acids (leucine 25, arginine 36, isoleucine 42, and glycine 59) contributes to DNA binding in the context of the N-terminal 123 amino acids of SB transposase, as indicated by electrophoretic mobility shift analysis, and to functional activity of the full-length transposase, as determined by a quantitative HeLa cell-based transposition assay. Moreover, we show that amino acid substitutions within either the putative oligomerization domain (L11A, L18A, L25A, and L32A) or the nuclear localization signal (K104A and R105A) severely impair its ability to mediate DNA transposition in mammalian cells. In contrast, each of 10 single amino acid changes within the bipartite DNA-binding domain is shown to greatly enhance SB's transpositional activity in mammalian cells. These hyperactive mutations functioned synergistically when combined and are shown to significantly improve transposase affinity for transposon end sequences. Finally, we show that enhanced DNA-binding activity results in improved cleavage kinetics, increased SB element mobilization from host cell chromosomes, and dramatically improved gene transfer capabilities of SB in vivo in mice. These studies provide important insights into vertebrate transposon biology and indicate that Sleeping Beauty can be readily improved for enhanced genetic research applications in mammals.

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