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

Abstract

Cyclic GMP (cGMP)-dependent protein kinase (protein kinase G [PKG]) is essential for microneme secretion, motility, invasion, and egress in apicomplexan parasites, However, the separate roles of two isoforms of the kinase that are expressed by some apicomplexans remain uncertain. Despite having identical regulatory and catalytic domains, PKGI is plasma membrane associated whereas PKGII is cytosolic in Toxoplasma gondii. To determine whether these isoforms are functionally distinct or redundant, we developed an auxin-inducible degron (AID) tagging system for conditional protein depletion in T. gondii. By combining AID regulation with genome editing strategies, we determined that PKGI is necessary and fully sufficient for PKG-dependent cellular processes. Conversely, PKGII is functionally insufficient and dispensable in the presence of PKGI. The difference in functionality mapped to the first 15 residues of PKGI, containing a myristoylated Gly residue at position 2 that is critical for membrane association and PKG function. Collectively, we have identified a novel requirement for cGMP signaling at the plasma membrane and developed a new system for examining essential proteins in T. gondii.

Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii

“…Set up the Q5 PCR reaction using pYFP-mAID-3HA, Floxed HXGPRT (Addgene #87259) (Brown et al ., 2017) or pYFP-AID-3HA, Floxed HXGPRT (Addgene #87260) (Long et al ., 2017b) plasmids as the template (Figure 2F). A standard 500 μl reaction consists of:…”

“…Incubation times needed to achieve complete protein knockdown will vary based on protein localization, stability, and abundance. As little as 15 min has been used to achieve complete knockdown of soluble cytosolic (m)AID-fusions such as YFP (Brown et al, 2017; Long et al, 2017b). Following knockdown, parasites can be examined for phenotypes directly, harvested for follow-up assays, or lysed for analysis of (m)AID-tagged protein levels.…”

“…T. gondii line RH TIR1-3FLAG (genotype: RHΔ hxgprt Δ ku80; TUB1:TIR1-3FLAG, SAG1:CAT ) (Brown et al ., 2017; Long et al ., 2017a)…”

“…Treatment with an auxin ( e.g., 3-indolacetic acid/IAA) activates the SCF TIR1 ubiquitin ligase complex which exclusively targets AID-tagged proteins for ubiquitin-dependent proteasomal degradation (Figure 1). We recently engineered an RHΔ hxgprt Δ ku80 line of T. gondii to stably express TIR1 from Oryza sativa (RH TIR1-3FLAG) (Brown et al ., 2017; Long et al ., 2017a). In this background, we were able to use CRISPR/Cas9 genome editing (Shen et al ., 2014; Sidik et al ., 2014; Shen et al ., 2017) to tag essential T. gondii genes of interest with AID-3HA or mini-AID(mAID)-3HA, regulate their expression with auxin, and identify phenotypes associated with their loss (Brown et al ., 2017; Long et al ., 2017a and 2017b).…”

Gliding motility powers invasion and egress in Apicomplexa

“…Several other proteins and molecules have been linked to signalling pathways that regulate the glideosome. cGMP-dependent protein kinase (PKG) triggers microneme secretion and parasite egress in a calcium-independent manner 153,[157][158][159] . Recently, phosphatidic acid has been identified as an important lipid mediator of microneme secretion that acts in parallel to calcium 160 .…”