Using the AKAR3-EV biosensor to assess Sch9p- and PKA-signalling in budding yeast.
Saccharomyces cerevisiae
FRET biosensor
PKA
Sch9
intracellular signalling
single-cell
Journal
FEMS yeast research
ISSN: 1567-1364
Titre abrégé: FEMS Yeast Res
Pays: England
ID NLM: 101085384
Informations de publication
Date de publication:
04 01 2023
04 01 2023
Historique:
received:
28
10
2022
revised:
01
05
2023
accepted:
10
05
2023
medline:
15
6
2023
pubmed:
13
5
2023
entrez:
12
5
2023
Statut:
ppublish
Résumé
Budding yeast uses the TORC1-Sch9p and cAMP-PKA signalling pathways to regulate adaptations to changing nutrient environments. Dynamic and single-cell measurements of the activity of these cascades will improve our understanding of the cellular adaptation of yeast. Here, we employed the AKAR3-EV biosensor developed for mammalian cells to measure the cellular phosphorylation status determined by Sch9p and PKA activity in budding yeast. Using various mutant strains and inhibitors, we show that AKAR3-EV measures the Sch9p- and PKA-dependent phosphorylation status in intact yeast cells. At the single-cell level, we found that the phosphorylation responses are homogenous for glucose, sucrose, and fructose, but heterogeneous for mannose. Cells that start to grow after a transition to mannose correspond to higher normalized Förster resonance energy transfer (FRET) levels, in line with the involvement of Sch9p and PKA pathways to stimulate growth-related processes. The Sch9p and PKA pathways have a relatively high affinity for glucose (K0.5 of 0.24 mM) under glucose-derepressed conditions. Lastly, steady-state FRET levels of AKAR3-EV seem to be independent of growth rates, suggesting that Sch9p- and PKA-dependent phosphorylation activities are transient responses to nutrient transitions. We believe that the AKAR3-EV sensor is an excellent addition to the biosensor arsenal for illuminating cellular adaptation in single yeast cells.
Identifiants
pubmed: 37173282
pii: 7161122
doi: 10.1093/femsyr/foad029
pmc: PMC10237333
pii:
doi:
Substances chimiques
Cyclic AMP-Dependent Protein Kinases
EC 2.7.11.11
Mannose
PHA4727WTP
Saccharomyces cerevisiae Proteins
0
Glucose
IY9XDZ35W2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.
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