Exploiting homologous recombination increases SATAY efficiency for loss- and gain-of-function screening

Michel, Agnès H.; Schie, Sabine van; Mosbach, Andreas; Scalliet, Gabriel; Kornmann, Benoı̂t

doi:10.1101/866483

Cited by 15 publications

(21 citation statements)

References 36 publications

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“…Transposition was induced by growing cells in galactose-containing medium for ~56 hours. Since transposition occurs predominantly in saturated phase (Michel et al , 2019), this generates millions of independent transposition events. The transposon libraries for a given genotype were then grown for several cycles in medium lacking tryptophan, in the Kennedy ON or Kennedy OFF conditions to i) select for cells that have successfully transposed and ii) to select against mutants that cannot grow in the rewired conditions.…”

Section: Resultsmentioning

confidence: 99%

“…Besides unique adaptations to specific conditions, rewired strains may have common strategies to deal with general phospholipid and membrane stress. To identify such genes, we compared the rewired libraries generated in this study, with two previously generated wild-type libraries (Fig 5A) (Michel et al , 2019). Expected changes in transposon insertion profiles were associated with technical differences in library generation, in the present and previous studies (e.g.…”

Section: Resultsmentioning

confidence: 99%

“…To identify the genetic components necessary to reroute lipid trafficking and handle lipid imbalances in the rewired strains, we performed genetic screens using SAturated Transposon Analysis in Yeast (SATAY) (Fig 3A) (Michel et al, 2019(Michel et al, , 2017. Briefly, we transformed rewired yeast cells with a plasmid containing a galactose-inducible transposase (TPase) and a transposon (TN) disrupting the TRP1 gene.…”

Section: A Transposon Mutagenesis Screen To Identify Genetic Components Essential For Survival Of Rewired Yeastmentioning

confidence: 99%

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Rewiring phospholipid biosynthesis reveals robustness in membrane homeostasis and uncovers lipid regulatory players

Peter

Schie

Cheung

et al. 2021

Preprint

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Intracellular transport of lipids by Lipid Transport Proteins (LTPs) is thought to work alongside vesicular transport to shuttle lipids from their place of synthesis to their destinations. Whereas many LTPs have been identified, it is largely unknown which routes and which LTPs a given lipid utilizes to navigate the multiple membranes of eukaryotic cells. The major and essential phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) can be produced by multiple pathways and, in the case of PE, also at multiple locations. Here, we present an approach in which we simplify and rewire yeast phospholipid synthesis by redirecting PE and PC synthesis reactions to distinct subcellular locations using chimeric enzymes fused to specific organelle targeting motifs. In rewired conditions, viability is expected to depend on homeostatic adaptation to the ensuing lipostatic perturbations and on efficient interorganelle lipid transport. We therefore performed genetic screens to identify factors involved in both of these processes. Among the candidates identified, we find genes linked to transcriptional regulation of lipid homeostasis, lipid metabolism and transport. In particular, we identify a requirement for Csf1 -an uncharacterized protein harboring a Chorein-N lipid transport domain- for survival under certain rewired conditions as well as lipidomic adaptation to cold, implicating Csf1 in interorganelle lipid transport and homeostatic adaptation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: A Transposon Mutagenesis Screen To Identify Genetic Components Essential For Survival Of Rewired Yeastmentioning

confidence: 99%

See 1 more Smart Citation

Rewiring phospholipid biosynthesis reveals robustness in membrane homeostasis and uncovers lipid regulatory players

Peter

Schie

Cheung

et al. 2021

Preprint

Self Cite

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“…The 57 million independent transposed clones library generated in the W303 background described in [ 86 ] was frozen for further usage following induction of the transposition and prior to regrowth in selective medium. The library (6 ml) was thawed to inoculate a 2 x 2L SD-ADE 2% glucose culture at OD 600 of 0.176.…”

Section: Methodsmentioning

confidence: 99%

Indole-3-acetic acid is a physiological inhibitor of TORC1 in yeast

et al. 2021

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Indole-3-acetic acid (IAA) is the most common, naturally occurring phytohormone that regulates cell division, differentiation, and senescence in plants. The capacity to synthesize IAA is also widespread among plant-associated bacterial and fungal species, which may use IAA as an effector molecule to define their relationships with plants or to coordinate their physiological behavior through cell-cell communication. Fungi, including many species that do not entertain a plant-associated life style, are also able to synthesize IAA, but the physiological role of IAA in these fungi has largely remained enigmatic. Interestingly, in this context, growth of the budding yeast Saccharomyces cerevisiae is sensitive to extracellular IAA. Here, we use a combination of various genetic approaches including chemical-genetic profiling, SAturated Transposon Analysis in Yeast (SATAY), and genetic epistasis analyses to identify the mode-of-action by which IAA inhibits growth in yeast. Surprisingly, these analyses pinpointed the target of rapamycin complex 1 (TORC1), a central regulator of eukaryotic cell growth, as the major growth-limiting target of IAA. Our biochemical analyses further demonstrate that IAA inhibits TORC1 both in vivo and in vitro. Intriguingly, we also show that yeast cells are able to synthesize IAA and specifically accumulate IAA upon entry into stationary phase. Our data therefore suggest that IAA contributes to proper entry of yeast cells into a quiescent state by acting as a metabolic inhibitor of TORC1.

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“…The strain’s six deletions made traditional synthetic genetic array analysis too difficult, so we used the saturated transposon analysis in yeast (SATAY) assay ( Michel et al. , 2017 , 2019 ). The SATAY approach utilizes massive libraries of transposon mutagenized strains.…”

Section: Introductionmentioning

confidence: 99%