scholarly journals Guanidine hydrochloride reactivates an ancient septin hetero-oligomer assembly pathway in budding yeast

2019 ◽  
Author(s):  
Courtney R. Johnson ◽  
Marc G. Steingesser ◽  
Andrew D. Weems ◽  
Anum Khan ◽  
Amy Gladfelter ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Small Molecule ◽  
Budding Yeast ◽  
Guanidine Hydrochloride ◽  
Fungal Species ◽  
Gtpase Activity ◽  
Wild Type ◽  
Cellular Functions ◽  
Fungal Evolution ◽  
Assembly Pathway

ABSTRACTSeptin proteins co-assemble into hetero-oligomers that polymerize into cytoskeletal filaments with a variety of cellular functions. In Saccharomyces cerevisiae, where septins were first discovered, five subunits comprise two species of septin hetero-octamers, Cdc11/Shs1–Cdc12–Cdc3–Cdc10– Cdc10–Cdc3–Cdc12–Cdc11/Shs1. Septins evolved from ancestral GTPases. We previously found evidence that slow GTPase activity by Cdc12 directs the choice of incorporation of Cdc11 vs Shs1 into septin complexes. It was unclear why many septins, including Cdc3, lack GTPase activity. We serendipitously discovered that the small molecule guanidine hydrochloride (GdnHCl) rescues septin function in cdc10 mutants by promoting assembly of non-native Cdc11/Shs1–Cdc12–Cdc3– Cdc3–Cdc12–Cdc11/Shs1 hexamers. We provide evidence that in S. cerevisiae Cdc3 guanidinium ion (Gdm) occupies the site of a “missing” Arg sidechain that is present in other fungal species in which (i) the Cdc3 subunit is an active GTPase and (ii) Cdc10-less hexamers co-exist with octamers in wild-type cells. These findings support a model in which Gdm reactivates a latent septin assembly pathway that was suppressed during fungal evolution in order to restrict assembly to hetero-octamers. Given that septin hexamers made natively in human cells also exclude Cdc10-like central subunits via homodimerization of an active GTPase, our results provide new mechanistic details that likely apply to septin assembly throughout phylogeny.

scholarly journals Guanidine hydrochloride reactivates an ancient septin hetero-oligomer assembly pathway in budding yeast

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Courtney R Johnson ◽  
Marc G Steingesser ◽  
Andrew D Weems ◽  
Anum Khan ◽  
Amy Gladfelter ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Guanidine Hydrochloride ◽  
Fungal Species ◽  
Side Chain ◽  
Gtpase Activity ◽  
Fungal Evolution ◽  
Assembly Pathway

Septin proteins evolved from ancestral GTPases and co-assemble into hetero-oligomers and cytoskeletal filaments. In Saccharomyces cerevisiae, five septins comprise two species of hetero-octamers, Cdc11/Shs1–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11/Shs1. Slow GTPase activity by Cdc12 directs the choice of incorporation of Cdc11 vs Shs1, but many septins, including Cdc3, lack GTPase activity. We serendipitously discovered that guanidine hydrochloride rescues septin function in cdc10 mutants by promoting assembly of non-native Cdc11/Shs1–Cdc12–Cdc3–Cdc3–Cdc12–Cdc11/Shs1 hexamers. We provide evidence that in S. cerevisiae Cdc3 guanidinium occupies the site of a ‘missing’ Arg side chain found in other fungal species where (i) the Cdc3 subunit is an active GTPase and (ii) Cdc10-less hexamers natively co-exist with octamers. We propose that guanidinium reactivates a latent septin assembly pathway that was suppressed during fungal evolution in order to restrict assembly to octamers. Since homodimerization by a GTPase-active human septin also creates hexamers that exclude Cdc10-like central subunits, our new mechanistic insights likely apply throughout phylogeny.

scholarly journals Novel Small-Molecule Inhibitors of RNA Polymerase III

2003 ◽  
Vol 2 (2) ◽  
pp. 256-264 ◽  
Author(s):  
Liping Wu ◽  
Jing Pan ◽  
Vala Thoroddsen ◽  
Deborah R. Wysong ◽  
Ronald K. Blackman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Rna Polymerase Iii ◽  
Wild Type ◽  
Biochemical Assays ◽  
Transcription In Vitro ◽  
Pol Iii

ABSTRACT A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.

scholarly journals Author response: Guanidine hydrochloride reactivates an ancient septin hetero-oligomer assembly pathway in budding yeast

2020 ◽  
Author(s):  
Courtney R Johnson ◽  
Marc G Steingesser ◽  
Andrew D Weems ◽  
Anum Khan ◽  
Amy Gladfelter ◽  
...  
Keyword(s):  
Budding Yeast ◽  
Guanidine Hydrochloride ◽  
Author Response ◽  
Assembly Pathway

scholarly journals IBD2 Encodes a Novel Component of the Bub2p-Dependent Spindle Checkpoint in the Budding Yeast Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 161 (2) ◽  
pp. 595-609
Author(s):  
Hyung-Seo Hwang ◽  
Kiwon Song
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Budding Yeast ◽  
Essential Gene ◽  
Spindle Checkpoint ◽  
Mitotic Arrest ◽  
Mitotic Exit ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Checkpoint Pathway

Abstract During mitosis, genomic integrity is maintained by the proper coordination of mitotic events through the spindle checkpoint. The bifurcated spindle checkpoint blocks cell cycle progression at metaphase by monitoring unattached kinetochores and inhibits mitotic exit in response to the incorrect orientation of the mitotic spindle. Bfa1p is a spindle checkpoint regulator of budding yeast in the Bub2p checkpoint pathway for proper mitotic exit. We have isolated a novel Bfa1p interacting protein named Ibd2p in the budding yeast Saccharomyces cerevisiae. We found that IBD2 (Inhibition of Bud Division 2) is not an essential gene but its deletion mutant proceeded through the cell cycle in the presence of microtubule-destabilizing drugs, thereby inducing a sharp decrease in viability. In addition, overexpression of Mps1p caused partial mitotic arrest in ibd2Δ as well as in bub2Δ, suggesting that IBD2 encodes a novel component of the spindle checkpoint downstream of MPS1. Overexpression of Ibd2p induced mitotic arrest with increased levels of Clb2p in wild type and mad2Δ, but not in deletion mutants of BUB2 and BFA1. Pds1p was also stabilized by the overexpression of Ibd2p in wild-type cells. The mitotic arrest defects observed in ibd2Δ in the presence of nocodazole were restored by additional copies of BUB2, BFA1, and CDC5, whereas an extra copy of IBD2 could not rescue the mitotic arrest defects of bub2Δ and bfa1Δ. The mitotic arrest defects of ibd2Δ were not recovered by MAD2, or vice versa. Analysis of the double mutant combinations ibd2Δmad2Δ, ibd2Δbub2Δ, and ibd2Δdyn1Δ showed that IBD2 belongs to the BUB2 epistasis group. Taken together, these data demonstrate that IBD2 encodes a novel component of the BUB2-dependent spindle checkpoint pathway that functions upstream of BUB2 and BFA1.

scholarly journals Aggregate Filamentous Growth Responses in Yeast

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Jacky Chow ◽  
Heather M. Dionne ◽  
Aditi Prabhakar ◽  
Amit Mehrotra ◽  
Jenn Somboonthum ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Signaling Pathways ◽  
Budding Yeast ◽  
Filamentous Growth ◽  
Fungal Species ◽  
Fungal Pathogenesis ◽  
Invasive Growth ◽  
Content Type ◽  
Secreted Enzymes

ABSTRACTMany fungal species, including pathogens, undergo a morphogenetic response called filamentous growth, where cells differentiate into a specialized cell type to promote nutrient foraging and surface colonization. Despite the fact that filamentous growth is required for virulence in some plant and animal pathogens, certain aspects of this behavior remain poorly understood. By examining filamentous growth in the budding yeastSaccharomyces cerevisiaeand the opportunistic pathogenCandida albicans, we identify responses where cells undergo filamentous growth in groups of cells or aggregates. InS. cerevisiae, aggregate invasive growth was regulated by signaling pathways that control normal filamentous growth. These pathways promoted aggregation in part by fostering aspects of microbial cooperation. For example, aggregate invasive growth required cellular contacts mediated by the flocculin Flo11p, which was produced at higher levels in aggregates than cells undergoing regular invasive growth. Aggregate invasive growth was also stimulated by secreted enzymes, like invertase, which produce metabolites that are shared among cells. Aggregate invasive growth was also induced by alcohols that promote density-dependent filamentous growth in yeast. Aggregate invasive growth also required highly polarized cell morphologies, which may affect the packing or organization of cells. A directed selection experiment for aggregating phenotypes uncovered roles for the fMAPK and RAS pathways, which indicates that these pathways play a general role in regulating aggregate-based responses in yeast. Our study extends the range of responses controlled by filamentation regulatory pathways and has implications in understanding aspects of fungal biology that may be relevant to fungal pathogenesis.IMPORTANCEFilamentous growth is a fungal morphogenetic response that is critical for virulence in some fungal species. Many aspects of filamentous growth remain poorly understood. We have identified an aspect of filamentous growth in the budding yeastSaccharomyces cerevisiaeand the human pathogenCandida albicanswhere cells behave collectively to invade surfaces in aggregates. These responses may reflect an extension of normal filamentous growth, as they share the same signaling pathways and effector processes. Aggregate responses may involve cooperation among individual cells, because aggregation was stimulated by cell adhesion molecules, secreted enzymes, and diffusible molecules that promote quorum sensing. Our study may provide insights into the genetic basis of collective cellular responses in fungi. The study may have ramifications in fungal pathogenesis, in situations where collective responses occur to promote virulence.

scholarly journals Layers of regulation of cell-cycle gene expression in the budding yeast Saccharomyces cerevisiae

2018 ◽  
Vol 29 (22) ◽  
pp. 2644-2655 ◽  
Author(s):  
Christina M. Kelliher ◽  
Matthew W. Foster ◽  
Francis C. Motta ◽  
Anastasia Deckard ◽  
Erik J. Soderblom ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Protein Expression ◽  
Ubiquitin Ligase ◽  
Budding Yeast ◽  
Cell Cycle Regulators ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Expression Levels ◽  
Mutant Cells

In the budding yeast Saccharomyces cerevisiae, transcription factors (TFs) regulate the periodic expression of many genes during the cell cycle, including gene products required for progression through cell-cycle events. Experimental evidence coupled with quantitative models suggests that a network of interconnected TFs is capable of regulating periodic genes over the cell cycle. Importantly, these dynamical models were built on transcriptomics data and assumed that TF protein levels and activity are directly correlated with mRNA abundance. To ask whether TF transcripts match protein expression levels as cells progress through the cell cycle, we applied a multiplexed targeted mass spectrometry approach (parallel reaction monitoring) to synchronized populations of cells. We found that protein expression of many TFs and cell-cycle regulators closely followed their respective mRNA transcript dynamics in cycling wild-type cells. Discordant mRNA/protein expression dynamics was also observed for a subset of cell-cycle TFs and for proteins targeted for degradation by E3 ubiquitin ligase complexes such as SCF (Skp1/Cul1/F-box) and APC/C (anaphase-promoting complex/cyclosome). We further profiled mutant cells lacking B-type cyclin/CDK activity ( clb1-6) where oscillations in ubiquitin ligase activity, cyclin/CDKs, and cell-cycle progression are halted. We found that a number of proteins were no longer periodically degraded in clb1-6 mutants compared with wild type, highlighting the importance of posttranscriptional regulation. Finally, the TF complexes responsible for activating G1/S transcription (SBF and MBF) were more constitutively expressed at the protein level than at periodic mRNA expression levels in both wild-type and mutant cells. This comprehensive investigation of cell-cycle regulators reveals that multiple layers of regulation (transcription, protein stability, and proteasome targeting) affect protein expression dynamics during the cell cycle.

Vcx1-D1 (M383I), the Vcx1 mutant with a calcineurin-independent vacuolar Ca2+/H+ exchanger activity, confers calcineurin-independent Mn2+ tolerance in Saccharomyces cerevisiae

2016 ◽  
Vol 62 (6) ◽  
pp. 475-484 ◽  
Author(s):  
Yunying Zhao ◽  
Huihui Xu ◽  
Yan Zhang ◽  
Linghuo Jiang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Signaling Pathway ◽  
Fluorescent Protein ◽  
Budding Yeast ◽  
Wild Type ◽  
Green Fluorescent ◽  
Calcineurin Signaling

The Vcx1-M1 mutant is known to confer calcineurin-dependent Mn2+ tolerance in budding yeast. Here, we demonstrate that another Vcx1 mutant, Vcx1-D1 with calcineurin-independent vacuolar Ca2+/H+ exchanger activity, confers calcineurin-independent Mn2+ tolerance. Unlike Vcx1-M1, the Mn2+ tolerance conferred by Vcx1-D1 is dependent on the presence of Pmr1 or Pmc1. The Pmr1-dependent Mn2+ tolerance of Vcx1-D1 requires the presence of calcineurin but not the functioning of the Ca2+/calcineurin signaling pathway. Similar to the wild-type Vcx1, C-terminally green fluorescent protein tagged Vcx1-D1 and Vcx1-M1 mutants localize to the endoplasmic reticulum instead of its normal vacuolar destination, but they remain functional in Ca2+ sensitivity and Mn2+ tolerance.

scholarly journals EXO1 Contributes to Telomere Maintenance in Both Telomerase-Proficient and Telomerase-Deficient Saccharomyces cerevisiae

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1651-1659 ◽  
Author(s):  
Alison A Bertuch ◽  
Victoria Lundblad
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Telomere Length ◽  
Budding Yeast ◽  
Telomere Maintenance ◽  
Wild Type ◽  
Rapid Loss ◽  
Telomere Shortening ◽  
Telomere Function

Abstract Previous work in budding yeast has indicated that telomeres are protected, at least in part, from the action of Exo1, which degrades the C-rich strand of partially uncapped telomeres. To explore this further, we examined the consequences of Exo1-mediated activity in strains that lacked Ku, telomerase, or both. Loss of Exo1 partially rescued the telomere length defect in a yku80Δ strain, demonstrating that exonuclease action can directly contribute to telomere shortening. The rapid loss of inviability displayed by a yku80Δ est2Δ strain was also partially alleviated by an exo1Δ mutation, further supporting the proposal that Exo1 is one target of the activities that normally protect wild-type telomeres. Conversely, however, Exo1 activity was also capable of enhancing telomere function and consequently cell proliferation, by contributing to a telomerase-independent pathway for telomere maintenance. The recovery of recombination-dependent survivors that arose in a yku80Δ est2Δ strain was partially dependent on Exo1 activity. Furthermore, the types of recombination events that facilitate telomerase-independent survival were influenced by Exo1 activity, in both est2Δ and yku80Δ est2Δ strains. These data demonstrate that Exo1 can make either positive or negative contributions to telomere function and cell viability, depending on whether telomerase or recombination is utilized to maintain telomere function.

scholarly journals Requirement of one functional RAS gene and inability of an oncogenic ras variant to mediate the glucose-induced cyclic AMP signal in the yeast Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (8) ◽  
pp. 3051-3057 ◽  
Author(s):  
K Mbonyi ◽  
M Beullens ◽  
K Detremerie ◽  
L Geerts ◽  
J M Thevelein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Transmission ◽  
Adenyl Cyclase ◽  
Gtpase Activity ◽  
Ras Proteins ◽  
Wild Type ◽  
Ras Protein ◽  
Oncogenic Ras ◽  
Camp Synthesis

Addition of glucose to Saccharomyces cerevisiae cells grown on a nonfermentable carbon source triggers a cyclic AMP (cAMP) signal, which induces a protein phosphorylation cascade. In a yeast strain lacking functional RAS1 and RAS2 genes and containing a bcy mutation to suppress the lethality of RAS deficiency, the cAMP signal was absent. Addition of dinitrophenol, which stimulates in vivo cAMP synthesis by lowering intracellular pH, also did not enhance the cAMP level. A bcy control strain, with functional RAS genes present, showed cAMP responses similar to those of a wild-type strain. In disruption mutants containing either a functional RAS1 gene or a functional RAS2 gene, the cAMP signal was not significantly different from the one in wild-type cells, indicating that RAS function cannot be a limiting factor for cAMP synthesis during induction of the signal. Compared with wild-type cells, the cAMP signal decreased in intensity with increasing temperature in a ras2 disruption mutant. When the mutant RAS2Val-19, which carries the equivalent of the human H-rasVal-12 oncogene, was grown under conditions in which RAS1 expression is repressed, the cAMP signal was absent. The oncogene product is known to be deficient in GTPase activity. However, the amino acid change at position 19 (or 12 in the corresponding human oncogene product) might also have other effects, such as abolishing receptor interaction. Such an additional effect probably provides a better explanation for the lack of signal transmission than the impaired GTPase activity. When the RAS2Val-19 mutant was grown under conditions in which RAS1 is expressed, the cAMP signal was present but significantly delayed compared with the signal in wild-type cells. This indicates that oncogenic RAS proteins inhibit normal functioning of wild-type RAS proteins in vivo and also that in spite of the presence of the RAS2(Val-19) oncogene, adenyl cyclase is not maximally stimulated in vivo. Expression of only the RAS(Val-19) gene product also prevented most of the stimulation of cAMP synthesis by dinitrophenol, indicating that lowered intracellular pH does not act directly on adenyl cyclase but on a step earlier in the activation pathway of the enzyme. The results obtained with the control bcy strain, the RAS2(Val-19) strain under conditions in which RAS1 is expressed, and with dinitrophenol show that the inability of the oncogene product to mediate the cAMP signal is not due to feedback inhibition by the high protein kinase activity in strains containing the RAS2(Val-19) oncogene. Hence, the present results show that the RAS protein in S. cerevisiae are involved in the transmission of the glucose-induced cAMP signal and that the oncogenic RAS protein is unable to act as a signal transducer. The RAS protein in S. cerevisiae apparently act similarly to the Gs proteins of mammalian adenyl cyclase, but instead of being involved in hormone signal transmission, they function in a nutrient-induced signal transmission pathway.

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