A functional genomic screen in Saccharomyces cerevisiae reveals divergent mechanisms of resistance to different alkylphosphocholine chemotherapeutic agents

Abstract The alkylphosphocholine (APC) class of antineoplastic and antiprotozoal drugs, such as edelfosine and miltefosine, are structural mimics of lyso-phosphatidylcholine (lyso-PC), and are inhibitory to the yeast Saccharomyces cerevisiae at low micromolar concentrations. Cytotoxic effects related to inhibition of phospholipid synthesis, induction of an unfolded protein response, inhibition of oxidative phosphorylation, and disruption of lipid rafts have been attributed to members of this drug class, however the molecular mechanisms of action of these drugs remain incompletely understood. Cytostatic and cytotoxic effects of the alkylphosphocholines exhibit variability with regard to chemical structure, leading to differences in effectiveness against different organisms or cell types. We now report the comprehensive identification of Saccharomyces cerevisiae titratable-essential gene and haploid non-essential gene deletion mutants that are resistant to the APC drug miltefosine (hexadecyl-O-phosphocholine). 58 strains out of ∼5600 tested displayed robust and reproducible resistance to miltefosine. This gene set was heavily enriched in functions associated with vesicular transport steps, especially those involving endocytosis and retrograde transport of endosome derived vesicles to the Golgi or vacuole, suggesting a role for these trafficking pathways in transport of miltefosine to potential sites of action in the endoplasmic reticulum (ER) and mitochondrion. In addition, we identified mutants with defects in phosphatidylinositol-4-phosphate synthesis (TetO::STT4) and hydrolysis (sac1Δ), an oxysterol binding protein homolog (osh2Δ), a number of ER resident proteins, and multiple components of the eisosome. These findings suggest that ER-plasma membrane contact sites and retrograde vesicle transport are involved in the interorganelle transport of lyso-PtdCho and related lyso-phospholipid-like analogs to their intracellular sites of cytotoxic activity.

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A functional genomic screen in Saccharomyces cerevisiae reveals divergent mechanisms of resistance to different alkylphosphocholine chemotherapeutic agents

10.1101/2020.10.16.343244 ◽

2020 ◽

Author(s):

Jacquelin M. Garcia ◽

Michael J. Schwabe ◽

Dennis R. Voelker ◽

Wayne R. Riekhof

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Mechanisms ◽

Essential Gene ◽

Retrograde Transport ◽

Cell Types ◽

Chemotherapeutic Agents ◽

Cytotoxic Effects ◽

Membrane Contact Sites ◽

Retrograde Vesicle Transport ◽

Sites Of Action

AbstractThe alkylphosphocholine (APC) class of antineoplastic and antiprotozoal drugs, such as edelfosine and miltefosine, are structural mimics of lyso-phosphatidylcholine (lyso-PC), and are inhibitory to the yeast Saccharomyces cerevisiae at low micromolar concentrations. Cytotoxic effects related to inhibition of phospholipid synthesis, induction of an unfolded protein response, inhibition of oxidative phosphorylation, and disruption of lipid rafts have been attributed to members of this drug class, however the molecular mechanisms of action of these drugs remain incompletely understood. Cytostatic and cytotoxic effects of the alkylphosphocholines exhibit variability with regard to chemical structure, leading to differences in effectiveness against different organisms or cell types. We now report the comprehensive identification of Saccharomyces cerevisiae titratable-essential gene and haploid non-essential gene deletion mutants that are resistant to the APC drug miltefosine (hexadecyl-O-phosphocholine). 58 strains out of ~5600 tested displayed robust and reproducible resistance to miltefosine. This gene set was heavily enriched in functions associated with vesicular transport steps, especially those involving endocytosis and retrograde transport of endosome derived vesicles to the Golgi or vacuole, suggesting a role for these trafficking pathways in transport of miltefosine to potential sites of action in the endoplasmic reticulum (ER) and mitochondrion. In addition, we identified mutants with defects in phosphatidylinositol-4-phosphate synthesis (TetO::STT4) and hydrolysis (sac1Δ), an oxysterol binding protein homolog (osh2Δ), a number of ER resident proteins, and multiple components of the eisosome. These findings suggest that ER-plasma membrane contact sites and retrograde vesicle transport are involved in the interorganelle transport of lyso-PtdCho and related lyso-phospholipid-like analogs to their intracellular sites of cytotoxic activity.

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Coupling DNA Replication and Spindle Function in Saccharomyces cerevisiae

Cells ◽

10.3390/cells10123359 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3359

Author(s):

Dimitris Liakopoulos

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Molecular Mechanisms ◽

Genome Duplication ◽

Yeast Cells ◽

Yeast Saccharomyces Cerevisiae ◽

Spindle Dynamics ◽

Eukaryotic Organisms ◽

Spindle Function

In the yeast Saccharomyces cerevisiae DNA replication and spindle assembly can overlap. Therefore, signaling mechanisms modulate spindle dynamics in order to ensure correct timing of chromosome segregation relative to genome duplication, especially when replication is incomplete or the DNA becomes damaged. This review focuses on the molecular mechanisms that coordinate DNA replication and spindle dynamics, as well as on the role of spindle-dependent forces in DNA repair. Understanding the coupling between genome duplication and spindle function in yeast cells can provide important insights into similar processes operating in other eukaryotic organisms, including humans.

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A presumptive helicase (MOT1 gene product) affects gene expression and is required for viability in the yeast Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.4.1879 ◽

1992 ◽

Vol 12 (4) ◽

pp. 1879-1892 ◽

Cited By ~ 98

Author(s):

J L Davis ◽

R Kunisawa ◽

J Thorner

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Essential Gene ◽

Single Gene ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Sequence Element ◽

Cis Acting ◽

Upstream Activating Sequence ◽

Identical Manner

Exposure of a haploid yeast cell to mating pheromone induces transcription of a set of genes. Induction is mediated through a cis-acting DNA sequence found upstream of all pheromone-responsive genes. Although the STE12 gene product binds specifically to this sequence element and is required for maximum levels of both basal and induced transcription, not all pheromone-responsive genes are regulated in an identical manner. To investigate whether additional factors may play a role in transcription of these genes, a genetic screen was used to identify mutants able to express pheromone-responsive genes constitutively in the absence of Ste12. In this way, we identified a recessive, single gene mutation (mot1, for modifier of transcription) which increases the basal level of expression of several, but not all, pheromone-responsive genes. The mot1-1 allele also relaxes the requirement for at least one other class of upstream activating sequence and enhances the expression of another gene not previously thought to be involved in the mating pathway. Cells carrying mot1-1 grow slowly at 30 degrees C and are inviable at 38 degrees C. The MOT1 gene was cloned by complementation of this temperature-sensitive lethality. Construction of a null allele confirmed that MOT1 is an essential gene. MOT1 residues on chromosome XVI and encodes a large protein of 1,867 amino acids which contains all seven of the conserved domains found in known and putative helicases. The product of MOT1 is strikingly homologous to the Saccharomyces cerevisiae SNF2/SW12 and RAD54 gene products over the entire helicase region.

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TMOD-38. PRODUCTION OF D-2-HYDROXYGLUTARATE IN SACCHAROMYCES CEREVISIAE LEADS TO GROWTH IMPAIRMENT AND DECREASED SURVIVAL

Neuro-Oncology ◽

10.1093/neuonc/noz175.1137 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi271-vi271

Author(s):

Sophie Fiola ◽

Eli Ganni ◽

Rita Lo ◽

Ka Yee Lok ◽

Elena Kuzmin ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Mechanisms ◽

De Novo ◽

Gene Interactions ◽

Low Grade ◽

Yeast Saccharomyces Cerevisiae ◽

Growth Impairment ◽

Knockout Strain ◽

Genomic Array ◽

Negative Gene

Abstract High levels of D-2-hydroxyglutarate (D2HG) are found in several types of cancers, most notably low grade gliomas (LGGs). The accumulation of D-2HG contributes to tumorigenesis through a variety of mechanisms including decreased utilization of oxidative phosphorylation and histone hypermethylation. The use of the budding yeast Saccharomyces cerevisiae as a model system to study cancer allows for faster, more efficient elucidation of various molecular mechanisms, including functional genomics via genomic array screening. S. cerevisiae encodes two homologs of the human D-2HG dehydrogenase: the mitochondrial Dld2 and cytosolic Dld3. We detected an increase in the production of D-2HG in the dld3∆ knockout strain by LC-MS. In addition, the dld3∆ knockout strain shows decreased survival and a growth impairment in glucose-containing liquid media. However, this strain did not show a significant growth impairment on glucose or glycerol-containing solid media. Using publicly available Synthetic Genomic Array (SGA) analysis data from TheCellMap.org, we investigated the top negative gene interactions for our dld3 knockout strain. GO analysis of these negative gene interactions showed enrichment of targets locating to the mitochondria, suggesting that the increase of 2-HG leads to mitochondrial impairment, consistent with previous observations in other models of LGGs. The top two targets of the SGA screen were mdm35, a mitochondrial interspace membrane protein involved in assembly of the mitochondrial respiratory chain complex and cdc8, a component of the de novo pyrimidine biosynthesis pathway. Taken together, these results suggest that the dld3∆ knockout strain is an appropriate model in which to study the D-2HG-driven changes that occur during tumorigenesis.

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Expression of human glutathione S-transferases in Saccharomyces cerevisiae confers resistance to the anticancer drugs adriamycin and chlorambucil

Biochemical Journal ◽

10.1042/bj2680309 ◽

1990 ◽

Vol 268 (2) ◽

pp. 309-315 ◽

Cited By ~ 90

Author(s):

S M Black ◽

J D Beggs ◽

J D Hayes ◽

A Bartoszek ◽

M Muramatsu ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Anticancer Drugs ◽

Direct Evidence ◽

Evolutionary Process ◽

Resistance Mechanisms ◽

Detoxification Enzymes ◽

Yeast Saccharomyces Cerevisiae ◽

Cytotoxic Effects ◽

Over Expression ◽

Glutathione S Transferases

Adaptation and resistance to chemicals in the environment is a critical part of the evolutionary process. As a result, a wide variety of defence systems that protect cells against chemical insult have evolved. Such chemical resistance mechanisms appear to play a central role in determining the sensitivity of human tumours to treatment with chemotherapeutic drugs. The glutathione S-transferases (GST) are important detoxification enzymes whose over-expression has been associated with drug-resistance. In order to evaluate this possibility we have expressed the human Alpha-class and Pi-class GST cDNAs that encode GST B1B1 and GST pi in the yeast Saccharomyces cerevisiae. The expression of GST B1B1 or GST pi resulted in a marked reduction in the cytotoxic effects of chlorambucil, a bifunctional alkylating agent, and an anthracycline, adriamycin. These data provide direct evidence that the over-expression of GST in cells can confer resistance to anticancer drugs.

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Insights into Responses to Extreme Environmental Conditions in Humans from Studies on Saccharomyces cerevisiae

Defence Science Journal ◽

10.14429/dsj.65.8651 ◽

2015 ◽

Vol 65 (6) ◽

pp. 444

Author(s):

Ramesh C. Meena ◽

Amitabha Chakrabarti

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Stress Response ◽

Gene Expression Data ◽

Molecular Mechanisms ◽

Multiple Stressors ◽

Expression Data ◽

Environmental Stress Response ◽

Yeast Saccharomyces Cerevisiae ◽

Pathways Analysis

<p>The versatility of the yeast experimental model has aided in innumerable ways in the understanding of fundamental cellular functions and has also contributed towards the elucidation of molecular mechanisms underlying several pathological conditions in humans. Genome-wide expression, functional, localization and interaction studies on the yeast Saccharomyces cerevisiae exposed to various stressors have made profound contributions towards the understanding of stress response pathways. Analysis of gene expression data from S. cerevisiae cells indicate that the expression of a common set of genes is altered upon exposure to all the stress conditions examined. This common response to multiple stressors is known as the Environmental stress response. Knowledge gained from studies on the yeast model has now become helpful in understanding stress response pathways and associated disease conditions in humans. Cross-species microarray experiments and analysis of data with ever improving computational methods has led to a better comparison of gene expression data between diverse organisms that include yeast and humans.</p>

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Retrograde Traffic Out of the Yeast Vacuole to the TGN Occurs via the Prevacuolar/Endosomal Compartment

The Journal of Cell Biology ◽

10.1083/jcb.142.3.651 ◽

1998 ◽

Vol 142 (3) ◽

pp. 651-663 ◽

Cited By ~ 89

Author(s):

Nia J. Bryant ◽

Robert C. Piper ◽

Lois S. Weisman ◽

Tom H. Stevens

Keyword(s):

Saccharomyces Cerevisiae ◽

Alkaline Phosphatase ◽

Amino Acid ◽

Retrograde Transport ◽

Carboxypeptidase Y ◽

Hybrid Protein ◽

Yeast Saccharomyces Cerevisiae ◽

Endosomal Compartment ◽

Sorting Motif ◽

Retrograde Traffic

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid–containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.

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The Role of Ancestral Duplicated Genes in Adaptation to Growth on Lactate, a Non-Fermentable Carbon Source for the Yeast Saccharomyces cerevisiae

International Journal of Molecular Sciences ◽

10.3390/ijms222212293 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12293

Author(s):

Florian Mattenberger ◽

Mario A. Fares ◽

Christina Toft ◽

Beatriz Sabater-Muñoz

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Source ◽

Molecular Mechanisms ◽

Functional Divergence ◽

Small Scale ◽

Whole Genome ◽

Duplicated Genes ◽

Acidic Stress ◽

Yeast Saccharomyces Cerevisiae ◽

Transcriptomic Response

The cell central metabolism has been shaped throughout evolutionary times when facing challenges from the availability of resources. In the budding yeast, Saccharomyces cerevisiae, a set of duplicated genes originating from an ancestral whole-genome and several coetaneous small-scale duplication events drive energy transfer through glucose metabolism as the main carbon source either by fermentation or respiration. These duplicates (~a third of the genome) have been dated back to approximately 100 MY, allowing for enough evolutionary time to diverge in both sequence and function. Gene duplication has been proposed as a molecular mechanism of biological innovation, maintaining balance between mutational robustness and evolvability of the system. However, some questions concerning the molecular mechanisms behind duplicated genes transcriptional plasticity and functional divergence remain unresolved. In this work we challenged S. cerevisiae to the use of lactic acid/lactate as the sole carbon source and performed a small adaptive laboratory evolution to this non-fermentative carbon source, determining phenotypic and transcriptomic changes. We observed growth adaptation to acidic stress, by reduction of growth rate and increase in biomass production, while the transcriptomic response was mainly driven by repression of the whole-genome duplicates, those implied in glycolysis and overexpression of ROS response. The contribution of several duplicated pairs to this carbon source switch and acidic stress is also discussed.

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CEP3 encodes a centromere protein of Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.128.5.749 ◽

1995 ◽

Vol 128 (5) ◽

pp. 749-760 ◽

Cited By ~ 48

Author(s):

A V Strunnikov ◽

J Kingsbury ◽

D Koshland

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Essential Gene ◽

Selection Procedure ◽

Permissive Temperature ◽

Yeast Saccharomyces Cerevisiae ◽

Centromere Protein ◽

Kinetochore Function ◽

Binding Component

We have designed a screen to identify mutants specifically affecting kinetochore function in the yeast Saccharomyces cerevisiae. The selection procedure was based on the generation of "synthetic acentric" minichromosomes. "Synthetic acentric" minichromosomes contain a centromere locus, but lack centromere activity due to combination of mutations in centromere DNA and in a chromosomal gene (CEP) encoding a putative centromere protein. Ten conditional lethal cep mutants were isolated, seven were found to be alleles of NDC10 (CEP2) encoding the 110-kD protein of yeast kinetochore. Three mutants defined a novel essential gene CEP3. The CEP3 product (Cep3p) is a 71-kD protein with a potential DNA-binding domain (binuclear Zn-cluster). At nonpermissive temperature the cep3 cells arrest with an undivided nucleus and a short mitotic spindle. At permissive temperature the cep3 cells are unable to support segregation of minichromosomes with mutations in the central part of element III of yeast centromere DNA. These minichromosomes, when isolated from cep3 cultures, fail to bind bovine microtubules in vitro. The sum of genetic, cytological and biochemical data lead us to suggest that the Cep3 protein is a DNA-binding component of yeast centromere. Molecular mass and sequence comparison confirm that Cep3p is the p64 component of centromere DNA binding complex Cbf3 (Lechner, 1994).

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Distinct Transcriptional Changes in Response to Patulin Underlie Toxin Biosorption Differences in Saccharomyces cerevisiae

Toxins ◽

10.3390/toxins11070400 ◽

2019 ◽

Vol 11 (7) ◽

pp. 400 ◽

Cited By ~ 4

Author(s):

Christian Oporto ◽

Carlos Villarroel ◽

Sebastián Tapia ◽

Verónica García ◽

Francisco Cubillos

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosome Biogenesis ◽

Molecular Mechanisms ◽

Protein Biosynthesis ◽

Redox Homeostasis ◽

Active Role ◽

Membrane Composition ◽

Yeast Saccharomyces Cerevisiae ◽

Significant Gene ◽

Membrane Components

Patulin (4-hydroxy-4H-furo[3,2c]pyran-2[6H]-one) is a mycotoxin produced by a suite of fungi species. Patulin is toxic to humans and is a sporadic contaminant in products that were made from fungi-infected fruits. The baker yeast Saccharomyces cerevisiae (S. cerevisiae) has been shown to decrease patulin levels likely by converting it to the less harmful E-ascladiol, yet this capacity is dependent on the strain utilized. In this study we show that four representative strains of different S. cerevisiae lineages differ in their ability to tolerate and decrease patulin levels in solution, demonstrating that some strains are better suitable for patulin biocontrol. Indeed, we tested the biocontrol capacities of the best patulin-reducer strain (WE) in contaminated apple juice and demonstrated their potential role as an efficient natural biocontrol solution. To investigate the mechanisms behind the differences between strains, we explored transcriptomic changes of the top (WE strain) and worst (WA strain) patulin-biocontroller strains after being exposed to this toxin. Large and significant gene expression differences were found between these two strains, the majority of which represented genes associated with protein biosynthesis, cell wall composition and redox homeostasis. Interestingly, the WE isolate exhibited an overrepresentation of up-regulated genes involved in membrane components, suggesting an active role of the membrane towards patulin detoxification. In contrast, WA upregulated genes were associated with RNA metabolism and ribosome biogenesis, suggesting a patulin impact upon transcription and translation activity. These results suggest that different genotypes of S. cerevisiae encounter different stresses from patulin toxicity and that different rates of detoxification of this toxin might be related with the plasma membrane composition. Altogether, our data demonstrates the different molecular mechanisms in S. cerevisiae strains withstanding patulin exposure and opens new avenues for the selection of new patulin biocontroller strains.

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