Inner Membrane Complex Proteins Research Articles

PP1 phosphatase controls both daughter cell formation and amylopectin levels in Toxoplasma gondii.

Virulence of apicomplexan parasites is based on their ability to divide rapidly to produce significant biomass. The regulation of their cell cycle is therefore key to their pathogenesis. Phosphorylation is a crucial posttranslational modification that regulates many aspects of the eukaryotic cell cycle. The phosphatase PP1 is known to play a major role in the phosphorylation balance in eukaryotes. We explored the role of TgPP1 during the cell cycle of the tachyzoite form of the apicomplexan parasite Toxoplasma gondii. Using a conditional mutant strain, we show that TgPP1 regulates many aspects of the cell cycle including the proper assembly of the daughter cells' inner membrane complex (IMC), the segregation of organelles, and nuclear division. Unexpectedly, depletion of TgPP1 also results in the accumulation of amylopectin, a storage polysaccharide that is usually found in the latent bradyzoite form of the parasite. Using transcriptomics and phospho-proteomics, we show that TgPP1 mainly acts through posttranslational mechanisms by dephosphorylating target proteins including IMC proteins. TgPP1 also dephosphorylates a protein bearing a starch-binding domain. Mutagenesis analysis reveals that the targeted phospho-sites are linked to the ability of the parasite to regulate amylopectin steady-state levels. Therefore, we show that TgPP1 has pleiotropic roles during the tachyzoite cell cycle regulation, but also regulates amylopectin accumulation.

BCC0 collaborates with IMC32 and IMC43 to form the Toxoplasma gondii essential daughter bud assembly complex.

Toxoplasma gondii divides by endodyogeny, in which two daughter buds are formed within the cytoplasm of the maternal cell using the inner membrane complex (IMC) as a scaffold. During endodyogeny, components of the IMC are synthesized and added sequentially to the nascent daughter buds in a tightly regulated manner. We previously showed that the early recruiting proteins IMC32 and IMC43 form an essential daughter bud assembly complex which lays the foundation of the daughter cell scaffold in T. gondii. In this study, we identify the essential, early recruiting IMC protein BCC0 as a third member of this complex by using IMC32 as bait in both proximity labeling and yeast two-hybrid screens. We demonstrate that BCC0's localization to daughter buds depends on the presence of both IMC32 and IMC43. Deletion analyses and functional complementation studies reveal that residues 701-877 of BCC0 are essential for both its localization and function and that residues 1-899 are sufficient for function despite minor mislocalization. Pairwise yeast two-hybrid assays additionally demonstrate that BCC0's essential domain binds to the coiled-coil region of IMC32 and that BCC0 and IMC43 do not directly interact. This data supports a model for complex assembly in which an IMC32-BCC0 subcomplex initially recruits to nascent buds via palmitoylation of IMC32 and is locked into the scaffold once bud elongation begins by IMC32 binding to IMC43. Together, this study dissects the organization and function of a complex of three early recruiting daughter proteins which are essential for the proper assembly of the IMC during endodyogeny.

Identification of IMC43, a novel IMC protein that collaborates with IMC32 to form an essential daughter bud assembly complex in Toxoplasma gondii.

The inner membrane complex (IMC) of Toxoplasma gondii is essential for all phases of the parasite's life cycle. One of its most critical roles is to act as a scaffold for the assembly of daughter buds during replication by endodyogeny. While many daughter IMC proteins have been identified, most are recruited after bud initiation and are not essential for parasite fitness. Here, we report the identification of IMC43, a novel daughter IMC protein that is recruited at the earliest stages of daughter bud initiation. Using an auxin-inducible degron system we show that depletion of IMC43 results in aberrant morphology, dysregulation of endodyogeny, and an extreme defect in replication. Deletion analyses reveal a region of IMC43 that plays a role in localization and a C-terminal domain that is essential for the protein's function. TurboID proximity labelling and a yeast two-hybrid screen using IMC43 as bait identify 30 candidate IMC43 binding partners. We investigate two of these: the essential daughter protein IMC32 and a novel daughter IMC protein we named IMC44. We show that IMC43 is responsible for regulating the localization of both IMC32 and IMC44 at specific stages of endodyogeny and that this regulation is dependent on the essential C-terminal domain of IMC43. Using pairwise yeast two-hybrid assays, we determine that this region is also sufficient for binding to both IMC32 and IMC44. As IMC43 and IMC32 are both essential proteins, this work reveals the existence of a bud assembly complex that forms the foundation of the daughter IMC during endodyogeny.

Inner membrane complex proteomics reveals a palmitoylation regulation critical for intraerythrocytic development of malaria parasite.

Malaria is caused by infection of the erythrocytes by the parasites Plasmodium. Inside the erythrocytes, the parasites multiply via schizogony, an unconventional cell division mode. The inner membrane complex (IMC), an organelle located beneath the parasite plasma membrane, serving as the platform for protein anchorage, is essential for schizogony. So far, the complete repertoire of IMC proteins and their localization determinants remain unclear. Here we used biotin ligase (TurboID)-based proximity labeling to compile the proteome of the schizont IMC of the rodent malaria parasite Plasmodium yoelii. In total, 300 TurboID-interacting proteins were identified. 18 of 21 selected candidates were confirmed to localize in the IMC, indicating good reliability. In light of the existing palmitome of Plasmodium falciparum, 83 proteins of the P. yoelii IMC proteome are potentially palmitoylated. We further identified DHHC2 as the major resident palmitoyl-acyl-transferase of the IMC. Depletion of DHHC2 led to defective schizont segmentation and growth arrest both in vitro and in vivo. DHHC2 was found to palmitoylate two critical IMC proteins CDPK1 and GAP45 for their IMC localization. In summary, this study reports an inventory of new IMC proteins and demonstrates a central role of DHHC2 in governing the IMC localization of proteins during the schizont development.

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

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single-celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity-dependent biotin identification (BioID)-based proteomics approach, using the established IMC marker protein Photosensitized INA-Labelled protein 1 (PhIL1) as bait in asexual blood-stage parasites. Subsequent mass spectrometry-based peptide identification revealed enrichment of 12 known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP-tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represents structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood-stage parasites.

Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes.

Male and female gametocytes are sexual precursor cells essential for mosquito transmission of malaria parasite. Differentiation of gametocytes into fertile gametes (known as gametogenesis) relies on the gender‐specific transcription program. How the parasites establish distinct repertoires of transcription in the male and female gametocytes remains largely unknown. Here, we report that an Apetala2 family transcription factor AP2‐O3 operates as a transcription repressor in the female gametocytes. AP2‐O3 is specifically expressed in the female gametocytes. AP2‐O3‐deficient parasites produce apparently normal female gametocytes. Nevertheless, these gametocytes fail to differentiate into fully fertile female gametes, leading to developmental arrest in fertilization and early development post‐fertilization. AP2‐O3 disruption causes massive upregulation of transcriptionally dormant male genes and simultaneously downregulation of highly transcribed female genes in the female gametocytes. AP2‐O3 targets a substantial proportion of the male genes by recognizing an 8‐base DNA motif. In addition, the maternal AP2‐O3 is removed after fertilization, which is required for the zygote to ookinete development. Therefore, the global transcriptional repression of the male genes in the female gametocytes is required for safeguarding female‐specific transcriptome and essential for the mosquito transmission of Plasmodium.

A protein palmitoylation cascade regulates microtubule cytoskeleton integrity in Plasmodium

Morphogenesis of many protozoans depends on a polarized establishment of cytoskeletal structures. In malaria‐causing parasites, this can be observed when a round zygote develops into an elongated motile ookinete within the mosquito stomach. This morphogenesis is mediated by the pellicle cytoskeletal structures, including the inner membrane complex (IMC) and the underlying subpellicular microtubules (SPMs). How the parasite maintains the IMC‐SPM connection and establishes a dome‐like structure of SPM to support cell elongation is unclear. Here, we show that palmitoylation of N‐terminal cysteines of two IMC proteins (ISP1/ISP3) regulates the IMC localization of ISP1/ISP3 and zygote‐to‐ookinete differentiation. Palmitoylation of ISP1/ISP3 is catalyzed by an IMC‐residing palmitoyl‐S‐acyl‐transferase (PAT) DHHC2. Surprisingly, DHHC2 undergoes self‐palmitoylation at C‐terminal cysteines via its PAT activity, which controls DHHC2 localization in IMC after zygote formation. IMC‐anchored ISP1 and ISP3 interact with microtubule component β‐tubulin, serving as tethers to maintain the proper structure of SPM during zygote elongation. This study identifies the first PAT–substrate pair in malaria parasites and uncovers a protein palmitoylation cascade regulating microtubule cytoskeleton.

A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex.

The Toxoplasma gondii inner membrane complex (IMC) is an important organelle involved in parasite motility and replication. The IMC resides beneath the parasite’s plasma membrane and is composed of both membrane and cytoskeletal components. Although the protein composition of the IMC is becoming better understood, the protein–protein associations that enable proper functioning of the organelle remain largely unknown. Determining protein interactions in the IMC cytoskeletal network is particularly challenging, as disrupting the cytoskeleton requires conditions that disrupt protein complexes. To circumvent this problem, we demonstrate the application of a photoreactive unnatural amino acid (UAA) crosslinking system to capture protein interactions in the native intracellular environment. In addition to identifying binding partners, the UAA approach maps the binding interface of the bait protein used for crosslinking, providing structural information of the interacting proteins. We apply this technology to the essential IMC protein ILP1 and demonstrate that distinct regions of its C-terminal coiled-coil domain crosslink to the alveolins IMC3 and IMC6, as well as IMC27. We also show that the IMC3 C-terminal domain and the IMC6 N-terminal domain are necessary for binding to ILP1, further mapping interactions between ILP1 and the cytoskeleton. Together, this study develops a new approach to study protein–protein interactions in Toxoplasma and provides the first insight into the architecture of the cytoskeletal network of the apicomplexan IMC.

Inner membrane complex 1l protein of Plasmodium falciparum links membrane lipids with cytoskeletal element ‘actin’ and its associated motor ‘myosin’

The inner membrane complex (IMC) is a defining feature of apicomplexans comprising of lipid and protein components involved in gliding motility and host cell invasion. Motility of Plasmodium parasites is accomplished by an actin and myosin based glideosome machinery situated between the parasite plasma membrane (PPM) and IMC. Here, we have studied in vivo expression and localization of a Plasmodium falciparum (Pf) IMC protein ‘PfIMC1l’ and characterized it functionally by using biochemical assays. We have identified cytoskeletal protein ‘actin’ and motor protein ‘myosin’ as novel binding partners of PfIMC1l, alongside its interaction with the lipids ‘cholesterol’ and ‘phosphatidyl-inositol 4, 5 bisphosphate’ (PIP2). While actin and myosin compete for interaction with PfIMC1l, actin and either of the lipids (cholesterol or PIP2) simultaneously bind PfIMC1l. Interestingly, PfIMC1l showed enhanced binding with actin in the presence of calcium ions, and displayed direct binding with calcium. Based on our in silico analysis and experimental data showing PfIMC1l-actin/myosin and PfIMC1l-lipid interactions, we propose that this protein may anchor the IMC membrane with the parasite gliding apparatus. Considering its binding with key proteins involved in motility viz. myosin and actin (with calcium dependence), we suggest that PfIMC1l may have a role in the locomotion of Plasmodium.

Differential Roles for Inner Membrane Complex Proteins across Toxoplasma gondii and Sarcocystis neurona Development.

The inner membrane complex (IMC) of apicomplexan parasites contains a network of intermediate filament-like proteins. The 14 alveolin domain-containing IMC proteins in Toxoplasma gondii fall into different groups defined by their distinct spatiotemporal dynamics during the internal budding process of tachyzoites. Here, we analyzed representatives of different IMC protein groups across all stages of the Toxoplasma life cycle and during Sarcocystis neurona asexual development. We found that across asexually dividing Toxoplasma stages, IMC7 is present exclusively in the mother's cytoskeleton, whereas IMC1 and IMC3 are both present in mother and daughter cytoskeletons (IMC3 is strongly enriched in daughter buds). In developing macro- and microgametocytes, IMC1 and -3 are absent, whereas IMC7 is lost in early microgametocytes but retained in macrogametocytes until late in their development. We found no roles for IMC proteins during meiosis and sporoblast formation. However, we observed that IMC1 and IMC3, but not IMC7, are present in sporozoites. Although the spatiotemporal pattern of IMC15 and IMC3 suggests orthologous functions in Sarcocystis, IMC7 may have functionally diverged in Sarcocystis merozoites. To functionally characterize IMC proteins, we knocked out IMC7, -12, -14, and -15 in Toxoplasma. IMC14 and -15 appear to be involved in switching between endodyogeny and endopolygeny. In addition, IMC7, -12, and -14, which are all recruited to the cytoskeleton outside cytokinesis, are critical for the structural integrity of extracellular tachyzoites. Altogether, stage- and development-specific roles for IMC proteins can be discerned, suggesting different niches for each IMC protein across the entire life cycle. IMPORTANCE The inner membrane complex (IMC) is a defining feature of apicomplexan parasites key to both their motility and unique cell division. To provide further insights into the IMC, we analyzed the dynamics and functions of representative alveolin domain-containing IMC proteins across developmental stages. Our work shows universal but distinct roles for IMC1, -3, and -7 during Toxoplasma asexual division but more specialized functions for these proteins during gametogenesis. In addition, we find that IMC15 is involved in daughter formation in both Toxoplasma and Sarcocystis. IMC14 and IMC15 function in limiting the number of Toxoplasma offspring per division. Furthermore, IMC7, -12, and -14, which are recruited in the G1 cell cycle stage, are required for stress resistance of extracellular tachyzoites. Thus, although the roles of the different IMC proteins appear to overlap, stage- and development-specific behaviors indicate that their functions are uniquely tailored to each life stage requirement.

Identification of Novel O-Linked Glycosylated Toxoplasma Proteins by Vicia villosa Lectin Chromatography.

Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins.

Pellicle formation in the malaria parasite.

ABSTRACTThe intraerythrocytic developmental cycle of Plasmodium falciparum is completed with the release of up to 32 invasive daughter cells, the merozoites, into the blood stream. Before release, the final step of merozoite development is the assembly of the cortical pellicle, a multi-layered membrane structure. This unique apicomplexan feature includes the inner membrane complex (IMC) and the parasite's plasma membrane. A dynamic ring structure, referred to as the basal complex, is part of the IMC and helps to divide organelles and abscises in the maturing daughter cells. Here, we analyze the dynamics of the basal complex of P. falciparum. We report on a novel transmembrane protein of the basal complex termed BTP1, which is specific to the genus Plasmodium. It colocalizes with the known basal complex marker protein MORN1 and shows distinct dynamics as well as localization when compared to other IMC proteins during schizogony. Using a parasite plasma membrane marker cell line, we correlate dynamics of the basal complex with the acquisition of the maternal membrane. We show that plasma membrane invagination and IMC propagation are interlinked during the final steps of cell division.

Novel components of the Toxoplasma inner membrane complex revealed by BioID.

ABSTRACTThe inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii.

Plasticity between MyoC- and MyoA-glideosomes: an example of functional compensation in Toxoplasma gondii invasion.

The glideosome is an actomyosin-based machinery that powers motility in Apicomplexa and participates in host cell invasion and egress from infected cells. The central component of the glideosome, myosin A (MyoA), is a motor recruited at the pellicle by the acylated gliding-associated protein GAP45. In Toxoplasma gondii, GAP45 also contributes to the cohesion of the pellicle, composed of the inner membrane complex (IMC) and the plasma membrane, during motor traction. GAP70 was previously identified as a paralog of GAP45 that is tailored to recruit MyoA at the apical cap in the coccidian subgroup of the Apicomplexa. A third member of this family, GAP80, is demonstrated here to assemble a new glideosome, which recruits the class XIV myosin C (MyoC) at the basal polar ring. MyoC shares the same myosin light chains as MyoA and also interacts with the integral IMC proteins GAP50 and GAP40. Moreover, a central component of this complex, the IMC-associated protein 1 (IAP1), acts as the key determinant for the restricted localization of MyoC to the posterior pole. Deletion of specific components of the MyoC-glideosome underscores the installation of compensatory mechanisms with components of the MyoA-glideosome. Conversely, removal of MyoA leads to the relocalization of MyoC along the pellicle and at the apical cap that accounts for residual invasion. The two glideosomes exhibit a considerable level of plasticity to ensure parasite survival.

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

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.

Targeted Disruption of TgPhIL1 in Toxoplasma gondii Results in Altered Parasite Morphology and Fitness

The inner membrane complex (IMC), a series of flattened vesicles at the periphery of apicomplexan parasites, is thought to be important for parasite shape, motility and replication, but few of the IMC proteins that function in these processes have been identified. TgPhIL1, a Toxoplasma gondii protein that was previously identified through photosensitized labeling with 5-[125I] iodonapthaline-1-azide, associates with the IMC and/or underlying cytoskeleton and is concentrated at the apical end of the parasite. Orthologs of TgPhIL1 are found in other apicomplexans, but the function of this conserved protein family is unknown. As a first step towards determining the function of TgPhIL1 and its orthologs, we generated a T. gondii parasite line in which the single copy of TgPhIL1 was disrupted by homologous recombination. The TgPhIL1 knockout parasites have a distinctly different morphology than wild-type parasites, and normal shape is restored in the knockout background after complementation with the wild-type allele. The knockout parasites are outcompeted in culture by parasites expressing functional TgPhIL1, and they generate a reduced parasite load in the spleen and liver of infected mice. These findings demonstrate a role for TgPhIL1 in the morphology, growth and fitness of T. gondii tachyzoites.

A family of intermediate filament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii

The intracellular protozoan parasite Toxoplasma gondii divides by a unique process of internal budding that involves the assembly of two daughter cells within the mother. The cytoskeleton of Toxoplasma, which is composed of microtubules associated with an inner membrane complex (IMC), has an important role in this process. The IMC, which is directly under the plasma membrane, contains a set of flattened membranous sacs lined on the cytoplasmic side by a network of filamentous proteins. This network contains a family of intermediate filament-like proteins or IMC proteins. In order to elucidate the division process, we have characterized a 14-member subfamily of Toxoplasma IMC proteins that share a repeat motif found in proteins associated with the cortical alveoli in all alveolates. By creating fluorescent protein fusion reporters for the family members we determined the spatiotemporal patterns of all 14 IMC proteins through tachyzoite development. This revealed several distinct distribution patterns and some provide the basis for novel structural models such as the assembly of certain family members into the basal complex. Furthermore we identified IMC15 as an early marker of budding and, lastly, the dynamic patterns observed throughout cytokinesis provide a timeline for daughter parasite development and division.

A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division

Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication.