Plasma Membrane Association by N-Acylation Governs PKG Function in Toxoplasma gondii

Area of Science:

• Cellular Biology
• Parasitology
• Molecular Biology

Background:

• Cyclic GMP (cGMP)-dependent protein kinase (PKG) is crucial for apicomplexan parasite functions like invasion and motility.
• Two PKG isoforms, PKG I (plasma membrane-associated) and PKG II (cytosolic), exist in Toxoplasma gondii, but their distinct roles are unclear.
• Understanding PKG isoform function is vital for targeting apicomplexan parasites, such as the pathogen Toxoplasma gondii.

Purpose of the Study:

• To investigate the functional differences and necessity of PKG I and PKG II isoforms in Toxoplasma gondii.
• To determine if PKG isoforms are functionally distinct or redundant in essential cellular processes.
• To establish a novel conditional protein depletion system for studying essential proteins in T. gondii.

Main Methods:

• Development of an auxin-inducible degron (AID) tagging system for conditional protein depletion in T. gondii.
• Combination of AID regulation with genome editing strategies to specifically degrade PKG isoforms.
• Analysis of PKG isoform functionality by assessing their necessity and sufficiency for PKG-dependent cellular processes.

Main Results:

• The plasma membrane-associated PKG I isoform was found to be necessary and fully sufficient for all PKG-dependent cellular processes.
• The cytosolic PKG II isoform was determined to be functionally insufficient and dispensable in the presence of PKG I.
• Functional differences were mapped to the N-terminal 15 residues of PKG I, with myristoylation at Gly2 critical for membrane association and function.

Conclusions:

• PKG I, localized at the plasma membrane, is the key functional isoform for essential PKG-mediated processes in T. gondii.
• PKG II plays a dispensable role, highlighting the specific importance of cGMP signaling at the parasite's plasma membrane.
• The developed AID system provides a powerful new tool for studying essential proteins in T. gondii and other apicomplexans.