N-terminal palmitoylation is required for Toxoplasma gondii HSP20 inner membrane complex localization

Abstract

Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis. Protein palmitoylation is known to play roles in signal transduction and in enhancing the hydrophobicity of proteins thus contributing to their membrane association. Global inhibition of protein palmitoylation has been shown to affect T. gondii physiology and invasion of the host cell. However, the proteins affected by this modification have been understudied. This paper shows that the small heat shock protein 20 from T. gondii (TgHSP20) is synthesized as a mature protein in the cytosol and is palmitoylated in three cysteine residues. However, its localization at the inner membrane complex (IMC) is dependent only on N-terminal palmitoylation. Absence or incomplete N-terminal palmitoylation causes TgHSP20 to partially accumulate in a membranous structure. Interestingly, TgHSP20 palmitoylation is not responsible for its interaction with the daughter cells IMCs. Together, our data describe the importance of palmitoylation in protein targeting to the IMC in T. gondii.

Identification of new palmitoylated proteins in Toxoplasma gondii

“…To avoid division by 0 we used 0.2 as an arbitrary value in those cases where no trace of the protein was detected in the HA- fraction. This list includes proteins already described to be palmitoylated (Table S1 highlighted in blue) such as HSP20 [12], GAP45 and AMA1 [15] and members of the ISP family [9], those predicted to be palmitoylated but without experimental evidence (Table S1 highlighted in grey) such as members of the ISC (IMC structure component), IMC (inner membrane complex) and AC (apical cap) families [32], and those that share homology to known S-acylated proteins from Plasmodium falciparum , a related organism, such as 14-3-3, Hsp70 and proteins of the CDPK family [33] (Table S1 highlighted in green). We also identified TGME49_213550 (TgDHHC 4), TGME49_249380 (TgDHHC13) and TGME49_278850 (TgDHHC2) which are DHHC S-acyl transferases [18].…”

“…We also included parasites expressing a Ty-tagged version of HSP20 as a control, since it has already been proved that HSP20 localization to the IMC is controlled by palmitoylation [12]. A clear change in HSP20 localization could be observed after 2-BP treatment, with the appearance of a characteristic “membranous” structure.…”

“…Similarly, the myosin light chain 2 (MLC2), a protein that is part of the motility system, has been reported to be anchored to the pellicle likely by N-terminal palmitoylation [11]. More recently, it has been demonstrated that the small heat shock protein 20 (TgHSP20) depends on N-terminal palmitoylation for proper IMC localization [12]. Furthermore, N-terminal myristoylation and palmitoylation are responsible for the attachment of the enzyme cGMP-dependent protein kinase (PKG) to the pellicle [13], and it has been demonstrated that an a rmadillo r epeat o nly protein (ARO) depends on myristoylation and palmitoylation for its localization on the cytosolic face of rhoptry membranes [14].…”

Emerging roles for protein S-palmitoylation in Toxoplasma biology

“…Three palmitoylated cysteine residues have been identified by radioactive palmitate labelling, however only the N-terminal pair of cysteine residues were found to be important for targeting, based on site-directed mutagenesis. Unexpectedly, acylation is not required for the interaction of HSP20 with the IMC of the developing daughter cells but is necessary to maintain the protein at the IMC of the mature parasites (De Napoli et al, 2013). The HSP20 orthologue in Plasmodium berghei has been reported to regulate sporozoite motility (Montagna et al, 2012).…”

Dynamic proteinS-palmitoylation mediates parasite life cycle progression and diverse mechanisms of virulence

“…IMC sub-compartment proteins ISP1–4 constitute a novel family of proteins specifically targeted to sub-domains in this organelle by multiple acylations in several apicomplexans (Beck et al , 2010, Fung et al , 2012, Poulin et al , 2013, Wetzel et al , 2015). In contrast, HSP20 only requires palmitoylation to localize to the IMC membrane (De Napoli et al , 2013). The IMC is remodeled throughout the Plasmodium life cycle (Figure 3A) and is required for the invasion of hepatocytes by sporozoites, erythrocytes by merozoites, and traversal of the peritrophic matrix and epithelial cells of the mosquito midgut by the ookinete (Harding and Meissner, 2014).…”