YEAST 2017

28th International Conference on Yeast Genetics and Molecular Biology (ICYGMB)

August 27 – September 1, 2017
Prague, Czech Republic


Paper ID: 267

Fructose-1,6-bisphosphate Activation of Ras and Nutrient Transceptor - eIF2B/eIF2 Interaction as Novel Mechanisms in Yeast Nutrient Signaling

Thevelein Johan1, Van Zeebroeck Griet1, Conrad Michaela1, Schothorst Joep1, Van Leemputte Frederik1, Zhang Zhiqiang1, Steyfkens Fenella1, Vanthienen Ward1, Fischer Baptiste2, Tsytlonok Maksym2, Tompa Peter2, Versées Wim2, Haesen Dorien3, Janssens Veerle3

1 VIB & KU Leuven (Belgium)
2 VIB & VUB (Belgium)
3 KU Leuven (Belgium)

ABSTRACT

The Ras-cAMP-PKA pathway in yeast mediates nutrient regulation of physiology, growth and development. Glucose addition to deprived cells causes a rapid spike in cAMP, which triggers activation of PKA. The cAMP spike is induced by combined action of the glucose-sensing GPCR, Gpr1, and activation of Ras by glycolytic breakdown of glucose, the mechanism of which has remained unclear. Re-addition of another essential nutrient, like nitrogen, phosphate, sulfate or a metal ion, to growth-arrested glucose-repressed cells deprived for such nutrient, triggers a similar rapid activation of the PKA pathway concomitant with upstart of growth. This activation is not mediated by an increase in the cAMP level, and although ample evidence has been obtained that starvation-induced high-affinity transporters function as transporter-receptors or transceptors in this nutrient activation process, the underlying molecular mechanism has remained unclear. We show that glycolytic activation of Ras is mediated by Fru1,6bisP, acting as metabolic messenger for coupling glycolytic flux to PKA. It is likely mediated by a conserved domain in yeast Cdc25 and Sos, its mammalian homolog, since biolayer interferometry measurements show that Fru1,6bisP stimulates dissociation of the pure Sos1/H-Ras complex with a KD of ±9 mM. This is in the physiological range of the cellular Fru1,6bisP level. Thermal shift assays confirm direct binding of Fru1,6bisP to pure Sos1. Glucose addition to mammalian cells also triggers rapid activation of Ras and its downstream targets MEK and ERK. Investigation of a possible role of protein synthesis initiation in PKA activation during nutrient-induced upstart of growth by addition of nitrogen, phosphate, sulfate or a metal ion to appropriately-starved cells, revealed that nutrient transceptors physically bind in vitro to eIF2B and eIF2 subunits. Bimolecular fluorescence complementation experiments with half-citrin tagged constructs confirm the interaction in vivo, which apparently occurs at multiple membranes. This suggests the concept of ‘startosome’, a membrane system controlling the start of protein synthesis. Differential nutrient transceptor-eIF2B/eIF2 interaction allows to design the first molecular model explaining how cells can detect with a common mechanism the absence of any single essential nutrient among the presence of all other essential nutrients, so as to downregulate protein synthesis and arrest cell cycle progression.

Keywords:
Nutrient signaling, Ras/cAMP/PKA pathway, Glycolytic activation of Ras, Yeast/mammalian conservation, Nutrient transceptors, Initiation of protein synthesis, eIF2B/eIF2
Presented as:
  Oral presentation [S3-1] in S3 Metabolism and stress response

Institute of Microbiology

YEAST 2017
28th International Conference on Yeast Genetics and Molecular Biology (ICYGMB)

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