Novel insights into the composition and function of theToxoplasmaIMC sutures

Abstract

Summary The Toxoplasma inner membrane complex (IMC) is a specialized organelle underlying the parasite’s plasma membrane that consists of flattened rectangular membrane sacs that are sutured together and positioned atop a supportive cytoskeleton. We have previously identified a novel class of proteins localizing to the transverse and longitudinal sutures of the IMC, which we named ISCs. Here we have used proximity-dependent biotin identification (BioID) at the sutures to better define the composition of this IMC subcompartment. Using ISC4 as bait, we demonstrate biotin-dependent labeling of the sutures and have uncovered two new ISCs. We also identified five new proteins that exclusively localize to the transverse sutures which we named TSCs, demonstrating that components of the IMC sutures consist of two groups, those that localize to the transverse and longitudinal sutures (ISCs) and those residing only in the transverse sutures (TSCs). In addition, we functionally analyze the ISC protein ISC3 and demonstrate that ISC3-null parasites have morphological defects and reduced fitness in vitro. Most importantly, Δisc3 parasites exhibit a complete loss of virulence in vivo. These studies expand the known composition of the IMC sutures and highlight the contribution of ISCs to the ability of the parasite to proliferate and cause disease.

Ancient MAPK ERK7 is regulated by an unusual inhibitory scaffold required for Toxoplasma apical complex biogenesis

“…AC9 (TGGT1_246950) was initially identified by proximity biotinylation as an apically localized interactor of the IMC suture component ISC4 (19), though AC9 function was not further investigated in the previous study. We were unable to obtain an AC9 knockout parasite strain using CRISPR/Cas9, thus we chose to assess its function using the auxin-inducible degron (AID) system (20,21).…”

“…We grew AC9 BioID parasites in biotin, lysed them in 1% Triton-X-100, and separated the cytoskeleton from solubilized membrane and cytosolic components by sedimentation at 14,000 × g. We then purified the biotinylated proteins using streptavidin resin, which were identified by LC-MS/MS (Supplemental Dataset S1). Our dataset was of high quality, as the top candidates were enriched in known apical cap proteins (12,19). In addition, our top hit was previously undescribed (TGGT1_292950).…”

Directed evolution of TurboID for efficient proximity labeling in living cells and organisms

“…By contrast, BioID is attractive because of the simplicity of its labeling protocol and non-toxic labeling conditionsonly biotin needs to be added to initiate tagging. These attributes have resulted in over 100 applications of BioID over the past 5 years, in cultured mammalian cells 5,8,9 , plant protoplasts 10 , parasites [11][12][13][14][15][16][17][18][19] , slime mold 20,21 , and mouse 22 . BioID has been used, for example, to map the protein composition of the centrosome-cilium interface 8 and the inhibitory postsynaptic region 22 , each with nanometer spatial specificity.The major disadvantage of BioID, however, is its slow kinetics, which necessitates labeling with biotin for 18-24 hours, and sometimes much longer 22 , to accumulate sufficient quantities of biotinylated material for proteomic analysis.…”

A novel dense granule protein, GRA41, regulates timing of egress and calcium sensitivity inToxoplasma gondii

“…Knockdown of the calcium-dependent kinase CDPK7 has previously been shown to disrupt normal division (Morlon-Guyot et al , 2014). Additionally, experiments to identify novel proteins in the inner membrane complex (IMC) of the parasite, which serves as the scaffold for daughter cell formation (Harding et al , 2014), identified a protein (ISC6, TgGT1_267620, ToxoDB.org), that localizes to the IMC and contains a C2 calcium binding domain (Chen et al , 2017), which could suggest a link between calcium and daughter parasite formation in T. gondii .…”