Combinatorial phosphorylation modulates the structure and function of the G protein γ subunit in yeast

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Signaling

Background:

• Intrinsically disordered regions (IDRs) regulate protein function through posttranslational modifications.
• N-terminal tails of G protein γ subunits are intrinsically disordered and influence G protein signaling.
• Combinatorial phosphorylation of IDRs fine-tunes cellular responses.

Purpose of the Study:

• To investigate combinatorial, multisite phosphorylation of the yeast Gγ subunit Ste18.
• To determine how specific stresses and stimuli affect Ste18 phosphorylation sites.
• To elucidate the impact of phosphorylation on Ste18 structure and downstream signaling.

Main Methods:

• Phosphoproteomic analysis of yeast cells under various stress conditions.
• Site-directed mutagenesis to probe phosphorylation site function.
• Biochemical assays to measure kinase activity and protein-protein interactions.
• Analysis of downstream mitogen-activated protein kinase (MAPK) pathway activation.

Main Results:

• Ste18's N-terminal IDR undergoes combinatorial phosphorylation at Ser7 and Ser3.
• Distinct stimuli (GPCR activation, osmotic stress, glucose, acid stress) induce site-specific phosphorylation.
• Phosphorylation at one site influences phosphorylation at the other, modulated by kinase activity.
• Phosphorylation alters Ste18 IDR structure and modulates Fus3 MAPK activation rate and amplitude.

Conclusions:

• Gγ subunits function as intrinsically disordered proteins regulated by combinatorial posttranslational modifications.
• Multisite phosphorylation of Ste18's IDR acts as a molecular switch, fine-tuning G protein signaling output.
• These findings reveal a novel mechanism for regulating signaling pathways through dynamic IDR phosphorylation.