Toxoplasma ISP4 is a central IMC Sub-compartment Protein whose localization depends on palmitoylation but not myristoylation

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095-1489, USA.
Molecular and Biochemical Parasitology (Impact Factor: 1.79). 05/2012; 184(2):99-108. DOI: 10.1016/j.molbiopara.2012.05.002
Source: PubMed


Apicomplexan parasites utilize a peripheral membrane system called the inner membrane complex (IMC) to facilitate host cell invasion and parasite replication. We recently identified a novel family of Toxoplasma IMC Sub-compartment Proteins (ISP1/2/3) that localize to sub-domains of the IMC using a targeting mechanism that is dependent on coordinated myristoylation and palmitoylation of a series of residues in the N-terminus of the protein. While the precise functions of the ISPs are unknown, deletion of ISP2 results in replication defects, suggesting that this family of proteins plays a role in daughter cell formation. Here we have characterized a fourth ISP family member (ISP4) and discovered that this protein localizes to the central IMC sub-compartment, similar to ISP2. Like ISP1/3, ISP4 is dispensable for the tachyzoite lytic cycle as the disruption of ISP4 does not produce any gross replication or growth defects. Surprisingly, targeting of ISP4 to the IMC membranes is dependent on residues predicted for palmitoylation but not myristoylation, setting its trafficking apart from the other ISP proteins and demonstrating distinct mechanisms of protein localization to the IMC membranes, even within a family of highly related proteins.

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Available from: Marc-Jan Gubbels, Sep 23, 2014
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    • "Newly assembled daughters, delimited by the IMC, ultimately emerge from the mother cell, picking up the maternal plasma membrane and sloughing off any residual maternal material (Sheffield and Melton, 1968). Many studies have focused on the cytoskeletal components of the IMC, and several Apicomplexan-specific IMC membrane proteins have been identified (Beck et al., 2010; Bullen et al., 2009; Fung et al., 2012), but our knowledge of alveolar membrane function remains incomplete (Harding and Meissner, 2014). Where does the IMC come from, and how is its assembly and turnover regulated? "
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    ABSTRACT: Unlike most cells, protozoa in the phylum apicomplexa divide by a distinctive process in which multiple daughters are assembled within the mother (schizogony, endodyogeny), using scaffolding known as the Inner Membrane Complex. The 'IMC' underlies the plasma membrane during interphase, but new daughters develop in the cytoplasm, as cytoskeletal filaments associate with flattened membrane cisternae (alveolae), which elongate rapidly to encapsulate subcellular organelles. Newly assembled daughters acquire their plasma membrane as they emerge from the mother, leaving behind vestiges of the maternal cell. While the maternal plasma membrane remains intact throughout this process, the maternal IMC disappears - is it degraded, or recycled to form the daughter IMC? Exploiting fluorescently tagged IMC markers, we have used live cell imaging, fluorescence photobleaching-recovery, and mEos2 photoactivation to monitor the dynamics of IMC biogenesis and turnover during Toxoplasma gondii tachyzoite replication. These studies reveal that formation of the T. gondii IMC involves two distinct steps: de novo assembly during daughter IMC elongation within the mother cell, followed by recycling of maternal IMC membranes after the emergence of daughters from the mother cell.
    Full-text · Article · Jun 2014 · Journal of Cell Science
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    • "In T. gondii, ISPs display a more hierarchical organisation whereas in Plasmodium such an organisation is not evident. For example, in T. gondii the presence of ISP1 at the apical cap excludes the other ISPs from this region (Beck et al., 2010; Fung et al., 2012), which is not seen in P. berghei. Only ISP1 is present in the anterior region and there is a clear boundary at the front and back, delineated by the presence of centrin-2. "
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    ABSTRACT: The phylum Apicomplexa comprises over 5000 intracellular protozoan parasites, including Plasmodium and Toxoplasma, that are clinically important pathogens affecting humans and livestock. Malaria parasites belonging to the genus Plasmodium possess a pellicle comprised of a plasmalemma and inner membrane complex (IMC), which is implicated in parasite motility and invasion. Using live cell imaging and reverse genetics in the rodent malaria model P. berghei, we localise two unique IMC sub-compartment proteins (ISPs) and examine their role in defining apical polarity during zygote (ookinete) development. We show that these proteins localise to the anterior apical end of the parasite where IMC organisation is initiated, and are expressed at all developmental stages, especially those that are invasive. Both ISP proteins are N-myristoylated, phosphorylated and membrane-bound. Gene disruption studies suggest that ISP1 is likely essential for parasite development, whereas ISP3 is not. However, an absence of ISP3 alters the apical localisation of ISP1 in all invasive stages including ookinetes and sporozoites, suggesting a coordinated function for these proteins in the organisation of apical polarity in the parasite.
    Full-text · Article · Nov 2013 · Biology Open
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    • "IMC Site-directed mutagenesis, N-terminal part fused to GFP Beck et al. (2010) TgISP4 IMC Site-directed mutagenesis Fung et al. (2012) TgHSP20 IMC Site-directed mutagenesis, [ 3 H] palmitate labelling De Napoli et al. (2013) TgIMC1, 4, 6, 9, 10, 11, 12, 13, 14, 15 IMC In silico prediction Anderson-White et al. (2011) TgMLC1 Pellicle Site-directed mutagenesis Frenal et al. (2010) TgGAP45 PM Site-directed mutagenesis Frenal et al. (2010) TgCDPK3 PM Site-directed mutagenesis Garrison et al. (2012) McCoy et al. (2012) Lourido et al. (2012) TgMLC2 PM Site-directed mutagenesis Polonais et al. (2011) Unpublished data PfGAP45 PM [ 3 H] palmitate labelling Rees-Channer et al. (2006) PfMTIP Pellicle P.f. schizont palmitome Jones et al. (2012) PfARO Rhoptry Site-directed mutagenesis, N-terminal part fused to GFP, ABE Cabrera et al. (2012) PfGAPM2, 3 IMC P.f. "
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    ABSTRACT: Post-translational modifications (PTMs) are refined, rapidly responsive and powerful ways to modulate protein function. Among PTMs, acylation is now emerging as a widespread modification exploited by eukaryotes, bacteria and viruses to control biological processes. Protein palmitoylation involves the attachment of palmitic acid, also known as hexadecanoic acid, to cysteine residues of integral and peripheral membrane proteins and increases their affinity for membranes. Importantly, similar to phosphorylation, palmitoylation is reversible and is becoming recognised as instrumental for the regulation of protein function by modulating protein interactions, stability, folding, trafficking and signalling. Palmitoylation appears to play a central role in the biology of the Apicomplexa, regulating critical processes such as host cell invasion which is vital for parasite survival and dissemination. The recent identification of over 400 palmitoylated proteins in Plasmodium falciparum erythrocytic stages illustrates the broad spread and impact of this modification on parasite biology. The main enzymes responsible for protein palmitoylation are multi-membrane protein S-acyl transferases (PATs) harbouring a catalytic Asp-His-His-Cys (DHHC) motif. A global functional analysis of the repertoire of PATs in Toxoplasma gondii and Plasmodium berghei has recently been performed. The essential nature of some of these enzymes illustrates the key roles played by this PTM in the corresponding substrates implicated in fundamental processes such as parasite motility and organelle biogenesis. Toward a better understanding of the depalmitoylation event, a protein with palmitoyl protein thioesterase (PPT) activity has been identified in T. gondii. TgPPT1/TgASH1 is the main target of specific acyl protein thioesterase inhibitors but is dispensable for parasite survival, suggesting the implication of other genes in depalmitoylation. Palmitoylation/depalmitoylation cycles are now emerging as potential novel regulatory networks and T. gondii represents a superb model organism in which to explore their significance.
    Full-text · Article · Oct 2013 · International journal for parasitology
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