Identification and Molecular Dissection of IMC32, a Conserved Toxoplasma Inner Membrane Complex Protein That Is Essential for Parasite Replication

Abstract

The inner membrane complex (IMC) is a unique organelle of apicomplexan parasites that plays critical roles in parasite motility, host cell invasion, and replication. Despite the common functions of the organelle, relatively few IMC proteins are conserved across the phylum and the precise roles of many IMC components remain to be characterized. Here, we identify a novel component of the Toxoplasma gondii IMC (IMC32) that localizes to the body portion of the IMC and is recruited to developing daughter buds early during endodyogeny. IMC32 is essential for parasite survival, as its conditional depletion results in a complete collapse of the IMC that is lethal to the parasite. We demonstrate that localization of IMC32 is dependent on both an N-terminal palmitoylation site and a series of C-terminal coiled-coil domains. Using deletion analyses and functional complementation, we show that two conserved regions within the C-terminal coiled-coil domains play critical roles in protein function during replication. Together, this work reveals an essential component of parasite replication that provides a novel target for therapeutic intervention of T. gondii and related apicomplexan parasites. IMPORTANCE The IMC is an important organelle that apicomplexan parasites use to maintain their intracellular lifestyle. While many IMC proteins have been identified, only a few central players that are essential for internal budding have been described and even fewer are conserved across the phylum. Here, we identify IMC32, a novel component of the Toxoplasma gondii IMC that localizes to very early daughter buds, indicating a role in the early stages of parasite replication. We then demonstrate that IMC32 is essential for parasite survival and pinpoint conserved regions within the protein that are important for membrane association and daughter cell formation. As IMC32 is unique to these parasites and not present in their mammalian hosts, it serves as a new target for the development of drugs that exclusively affect these important intracellular pathogens.

Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum

“…Within the top 58 enriched proteins that show a mean log 2 fold change >1 in the second elution steps are 12 known IMC proteins (PhIL1 (Parkyn Schneider et al, 2017; Saini et al, 2017), CINCH (Rudlaff et al, 2019), GAPM1‐3 (Bullen et al, 2009; Kono et al, 2012), ALV4/IMC1g (Hu et al, 2010), ALV5/IMC1c (Hu et al, 2010), IMC1f (Al‐Khattaf, Tremp, & Dessens, 2015), GAP45 (Baum et al, 2006), PF3D7_1345600 (Kono et al, 2012), BCP1 (Rudlaff et al, 2019) and ISP3 (Kono et al, 2012)) comprising approximately one quarter of all known IMC proteins (Ferreira et al, 2021), while another nine IMC proteins or homologues of T . gondii IMC proteins are among the proteins showing a mean log2FC >1 in the first elutions (IMC1m (Al‐Khattaf et al, 2015; Gao et al, 2018), ALV2 (Kono et al, 2012; Tremp, Al‐Khattaf, & Dessens, 2014), ALV1 (Al‐Khattaf, Tremp, El‐Houderi, & Dessens, 2017; Khater, Sinden, & Dessens, 2004), Pf IMC32 (Torres, Pasquarelli, Back, Moon, & Bradley, 2021), Pf ISC1 (Chen et al, 2015), ALV7 (Gao et al, 2018; Kono et al, 2012), Coronin (Bane et al, 2016), DHHC1 (Wetzel et al, 2015) and PIP1 (Parkyn Schneider et al, 2017). Additionally, the BioID‐based protein identification revealed with PPP8 a protein, that was previously identified using a CINCH immunoprecipitation (IP) approach (Rudlaff et al, 2019).…”

A Novel Toxoplasma Inner Membrane Complex Suture-Associated Protein Regulates Suture Protein Targeting and Colocalizes with Membrane Trafficking Machinery

“…Since the spotted localization of TGGT1_202220 differs from the peripheral localization of most IMC proteins, we examined structures associated with the IMC, including the cortical microtubules, apical annuli, and IMC sutures using smOLLAS (spaghetti monster Escherichia coli OmpF linker and mouse langerin fusion sequence)-tagged TGGT1_202220 ( 21 ). While we did not observe colocalization of the puncta with the microtubules or apical annuli ( Fig.…”

“…4A and B ), ISAP1 lacks homology to known proteins or domains that would provide a clue to its function. We have previously shown the importance of CC domains in the early daughter protein IMC32 and the IMC cytoskeletal protein ILP1 ( 21 , 24 ). To determine if the ISAP1 CC domains are important for targeting or function, we deleted each of the domains individually or together and expressed the deletion constructs in Δ isap1 parasites ( Fig.…”

A novel Toxoplasma IMC sutures-associated protein regulates suture protein targeting and colocalizes with membrane trafficking machinery

“…CCs are structural motifs that consist of alpha helices that are coiled together and often function in oligomerization or protein-protein interactions (18). We have previously shown that CCs play important roles in the IMC for the cytoskeletal protein ILP1 and the early daughter membrane protein IMC32, and these motifs are also frequently present in the constituents of the conoid and apical annuli in T. gondii (15,17,23,24). Surprisingly, deletion of the ISAP1 CCs individually or together demonstrated that they are dispensable for localization and function.…”

“…4A, B), ISAP1 lacks homology to known proteins or domains that would provide a clue towards its function. We have previously shown the importance of CC domains in the early daughter protein IMC32 and the IMC cytoskeletal protein ILP1 (23,24). To determine if the ISAP1 CC domains are important for targeting or function, we deleted each of the domains individually or together and expressed the deletion constructs in ∆isap1 parasites (Fig.…”