scholarly journals Systematic Identification of Protein Targets of Sub5 Using Saccharomyces cerevisiae Proteome Microarrays

2021 ◽  
Vol 22 (2) ◽  
pp. 760
Author(s):  
Pramod Shah ◽  
Chien-Sheng Chen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Filament ◽  
Interaction Network ◽  
Protein Domain ◽  
Actin Binding ◽  
Binding Motif ◽  
Systematic Analysis ◽  
Protein Targets ◽  
String Analysis ◽  
Go Enrichment

Antimicrobial peptides (AMPs) are intensively studied in terms of alternative drugs. Sub5 is a synthetic 12-mer AMP with substitutions of five amino acids of bactenecin 2A (Bac2A), a linear-ized bactenecin variant of bovine. Sub5 is highly effective against fungi with an ability to trans-locate cell membrane, but its targets are unknown. Systematic analysis of Sub5 targets will facil-itate our understanding on its mechanism of action. In this study, we used high-throughput Saccharomyces cerevisiae proteome microarrays to explore the potential protein targets of Sub5. The screening results showed 128 potential protein targets of Sub5. Bioinformatics analysis of protein targets of Sub5 revealed significant gene ontology (GO) enrichment in actin related pro-cess of “actin filament-based process”, “actin filament organization”, “actin cortical patch or-ganization”, regulation of “actin filament bundle assembly”. Moreover, the other enriched cat-egories in GO enrichment mostly contained actin associate proteins. In total, 11 actin-associated proteins were identified in the protein targets of Sub5. Protein family (PFAM) enrichment anal-ysis shows protein domain enriched in actin binding, i.e., “Cytoskeletal-regulatory complex EF hand (helix E-loop-helix F motif)”. Being consistent with GO analysis, Search Tool for the Re-trieval of Interacting Genes/Proteins (STRING) analysis of the protein targets of Sub5 showed ac-tin network with involvement of 15 protein targets. Along with actin-network, STRING analysis showed protein–protein interaction network in ribonucleoprotein, transcription and translation, chromosome, histone, and ubiquitin related, DNA repair, and chaperone. Multiple Expression motifs for Motif Elicitation (MEME) suite provided a consensus binding motif of [ED][ED]EEE[ED][ED][ED][ED][ED], in total of 75 protein targets of Sub5. This motif was present in 9 out of 15 actin-related proteins identified among protein targets of Sub5.

scholarly journals Protein disorder reduced in Saccharomyces cerevisiae to survive heat shock

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 1222
Author(s):  
Esmeralda Vicedo ◽  
Zofia Gasik ◽  
Yu-An Dong ◽  
Tatyana Goldberg ◽  
Burkhard Rost
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Interaction Network ◽  
Simple Algorithm ◽  
Protein Disorder ◽  
Differential Analysis ◽  
Protein Protein Interaction ◽  
Go Enrichment ◽  
Chromosome Iii ◽  
Shock Proteins

Recent experiments established that a culture of Saccharomyces cerevisiae (baker’s yeast) survives sudden high temperatures by specifically duplicating the entire chromosome III and two chromosomal fragments (from IV and XII). Heat shock proteins (HSPs) are not significantly over-abundant in the duplication. In contrast, we suggest a simple algorithm to “postdict” the experimental results: Find a small enough chromosome with minimal protein disorder and duplicate this region. This algorithm largely explains all observed duplications. In particular, all regions duplicated in the experiment reduced the overall content of protein disorder. The differential analysis of the functional makeup of the duplication remained inconclusive. Gene Ontology (GO) enrichment suggested over-representation in processes related to reproduction and nutrient uptake. Analyzing the protein-protein interaction network (PPI) revealed that few network-central proteins were duplicated. The predictive hypothesis hinges upon the concept of reducing proteins with long regions of disorder in order to become less sensitive to heat shock attack.

scholarly journals Actin filaments in yeast are unstable in the absence of capping protein or fimbrin.

1995 ◽  
Vol 131 (6) ◽  
pp. 1483-1493 ◽  
Author(s):  
T S Karpova ◽  
K Tatchell ◽  
J A Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Filament ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
Capping Protein ◽  
Filament Assembly

Many actin-binding proteins affect filament assembly in vitro and localize with actin in vivo, but how their molecular actions contribute to filament assembly in vivo is not understood well. We report here that capping protein (CP) and fimbrin are both important for actin filament assembly in vivo in Saccharomyces cerevisiae, based on finding decreased actin filament assembly in CP and fimbrin mutants. We have also identified mutations in actin that enhance the CP phenotype and find that those mutants also have decreased actin filament assembly in vivo. In vitro, actin purified from some of these mutants is defective in polymerization or binding fimbrin. These findings support the conclusion that CP acts to stabilize actin filaments in vivo. This conclusion is particularly remarkable because it is the opposite of the conclusion drawn from recent studies in Dictyostelium (Hug, C., P.Y. Jay, I. Reddy, J.G. McNally, P.C. Bridgman, E.L. Elson, and J.A. Cooper. 1995. Cell. 81:591-600). In addition, we find that the unpolymerized pool of actin in yeast is very small relative to that found in higher cells, which suggests that actin filament assembly is less dynamic in yeast than higher cells.

scholarly journals The Sla2p Talin Domain Plays a Role in Endocytosis in Saccharomyces cerevisiae

Genetics ◽  
2003 ◽  
Vol 165 (4) ◽  
pp. 1661-1674
Author(s):  
Jennifer J Baggett ◽  
Katharine E D'Aquino ◽  
Beverly Wendland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutant Strain ◽  
Genetic Interactions ◽  
Actin Binding ◽  
Binding Motif ◽  
Truncated Protein ◽  
Genes Encoding ◽  
Quadruple Mutant ◽  
Clathrin Adaptor ◽  
Multicellular Organisms

Abstract Clathrin-binding adaptors play critical roles for endocytosis in multicellular organisms, but their roles in budding yeast have remained unclear. To address this question, we created a quadruple mutant yeast strain lacking the genes encoding the candidate clathrin adaptors Yap1801p, Yap1802p, and Ent2p and containing a truncated version of Ent1p, Ent1ΔCBMp, missing its clathrin-binding motif. This strain was viable and competent for endocytosis, suggesting the existence of other redundant adaptor-like factors. To identify these factors, we mutagenized the quadruple clathrin adaptor mutant strain and selected cells that were viable in the presence of full-length Ent1p, but inviable with only Ent1ΔCBMp; these strains were named Rcb (requires clathrin binding). One mutant strain, rcb432, contained a mutation in SLA2 that resulted in lower levels of a truncated protein lacking the F-actin binding talin homology domain. Analyses of this sla2 mutant showed that the talin homology domain is required for endocytosis at elevated temperature, that SLA2 exhibits genetic interactions with both ENT1 and ENT2, and that the clathrin adaptors and Sla2p together regulate the actin cytoskeleton and revealed conditions under which Yap1801p and Yap1802p contribute to viability. Together, our data support the view that Sla2p is an adaptor that links actin to clathrin and endocytosis.

scholarly journals The interfaces of actin and Acanthamoeba actobindin. Identification of a new actin-binding motif

1991 ◽  
Vol 266 (23) ◽  
pp. 15427-15431 ◽  
Author(s):  
K. Vancompernolle ◽  
J. Vandekerckhove ◽  
M.R. Bubb ◽  
E.D. Korn
Keyword(s):  
Actin Binding ◽  
Binding Motif

scholarly journals The Putative RNA Helicase Dbp6p Functionally Interacts With Rpl3p, Nop8p and the Novel trans-acting Factor Rsa3p During Biogenesis of 60S Ribosomal Subunits in Saccharomyces cerevisiae

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1687-1699
Author(s):  
Jesús de la Cruz ◽  
Thierry Lacombe ◽  
Olivier Deloche ◽  
Patrick Linder ◽  
Dieter Kressler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosome Biogenesis ◽  
Null Allele ◽  
Ribosomal Subunit ◽  
Interaction Network ◽  
The Novel ◽  
Rna Helicases ◽  
Ribosomal Subunits ◽  
Synthetic Lethal ◽  
Synthetically Lethal

Abstract Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele.

Characterization of the human nebulette gene: a polymorphism in an actin-binding motif is associated with nonfamilial idiopathic dilated cardiomyopathy

2000 ◽  
Vol 107 (5) ◽  
pp. 440-451 ◽  
Author(s):  
Takuro Arimura ◽  
Takeyuki Nakamura ◽  
Shitoshi Hiroi ◽  
Manatsu Satoh ◽  
Megumi Takahashi ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Idiopathic Dilated Cardiomyopathy ◽  
Actin Binding ◽  
Binding Motif

scholarly journals Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding

2008 ◽  
Vol 183 (5) ◽  
pp. 865-879 ◽  
Author(s):  
Christian Frantz ◽  
Gabriela Barreiro ◽  
Laura Dominguez ◽  
Xiaoming Chen ◽  
Robert Eddy ◽  
...  
Keyword(s):  
Actin Filament ◽  
Structural Changes ◽  
Ph Sensor ◽  
Ph Sensitive ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Filament Dynamics ◽  
Ph Dependent ◽  
Dynamics Simulations

Newly generated actin free barbed ends at the front of motile cells provide sites for actin filament assembly driving membrane protrusion. Growth factors induce a rapid biphasic increase in actin free barbed ends, and we found both phases absent in fibroblasts lacking H+ efflux by the Na-H exchanger NHE1. The first phase is restored by expression of mutant cofilin-H133A but not unphosphorylated cofilin-S3A. Constant pH molecular dynamics simulations and nuclear magnetic resonance (NMR) reveal pH-sensitive structural changes in the cofilin C-terminal filamentous actin binding site dependent on His133. However, cofilin-H133A retains pH-sensitive changes in NMR spectra and severing activity in vitro, which suggests that it has a more complex behavior in cells. Cofilin activity is inhibited by phosphoinositide binding, and we found that phosphoinositide binding is pH-dependent for wild-type cofilin, with decreased binding at a higher pH. In contrast, phosphoinositide binding by cofilin-H133A is attenuated and pH insensitive. These data suggest a molecular mechanism whereby cofilin acts as a pH sensor to mediate a pH-dependent actin filament dynamics.

scholarly journals Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

2014 ◽  
Vol 42 (15) ◽  
pp. 9838-9853 ◽  
Author(s):  
Saeed Kaboli ◽  
Takuya Yamakawa ◽  
Keisuke Sunada ◽  
Tao Takagaki ◽  
Yu Sasano ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Interaction Network ◽  
Essential Genes ◽  
Genetic Interactions ◽  
Lethal Gene ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale

Abstract Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

scholarly journals Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation.

1992 ◽  
Vol 118 (3) ◽  
pp. 561-571 ◽  
Author(s):  
S Chowdhury ◽  
K W Smith ◽  
M C Gustin
Keyword(s):  
Osmotic Stress ◽  
Actin Filament ◽  
Physiological Process ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Yeast Saccharomyces Cerevisiae ◽  
Rhodamine Phalloidin ◽  
Diploid Cells

In the yeast Saccharomyces cerevisiae, actin filaments function to direct cell growth to the emerging bud. Yeast has a single essential actin gene, ACT1. Diploid cells containing a single copy of ACT1 are osmosensitive (Osms), i.e., they fail to grow in high osmolarity media (D. Shortle, unpublished observations cited by Novick, P., and D. Botstein. 1985. Cell. 40:415-426). This phenotype suggests that an underlying physiological process involving actin is osmosensitive. Here, we demonstrate that this physiological process is a rapid and reversible change in actin filament organization in cells exposed to osmotic stress. Filamentous actin was stained using rhodamine phalloidin. Increasing external osmolarity caused a rapid loss of actin filament cables, followed by a slower redistribution of cortical actin filament patches. In the recovery phase, cables and patches were restored to their original levels and locations. Strains containing an act1-1 mutation are both Osms and temperature-sensitive (Ts) (Novick and Botstein, 1985). To identify genes whose products functionally interact with actin in cellular responses to osmotic stress, we have isolated extragenic suppressors which revert only the Osms but not the Ts phenotype of an act1-1 mutant. These suppressors identify three genes, RAH1-RAH3. Morphological and genetic properties of a dominant suppressor mutation suggest that the product of the wild-type allele, RAH3+, is an actin-binding protein that interacts with actin to allow reassembly of the cytoskeleton following osmotic stress.

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