Aldolase Forms a Bridge between Cell Surface Adhesins and the Actin Cytoskeleton in Apicomplexan Parasites  Travis J. Jewett, L.David Sibley  Molecular Cell  Volume 11, Issue 4, Pages 885-894 (April 2003) DOI: 10.1016/S1097-2765(03)00113-8

Figure 1 Conserved Features of TRAP Family Proteins (A) The Toxoplasma MIC2 (TgMIC2) and Plasmodium TRAP (PfTRAP) proteins are type 1 transmembrane proteins comprised of extracellular adhesive domains (integrin-like A domain and thrombospondin type I repeat [TSP-1]), a transmembrane region (TM), and cytoplasmic domain (C-dom). (B) Alignment of the cytoplasmic domains of TRAP family proteins. Conserved tryptophan and glutamic acid residues are boxed. P. falciparum thrombospondin-related anonymous protein (PfTRAP, accession number AAA29778), T. gondii microneme protein 2 (TgMIC2, accession number AAB63303), T. gondii microneme protein 6 (TgMIC6, accession number AAD28185), P. falciparum circumsporozoite protein (PfCTRP, accession number CAB38978), Eimeria tenella microneme protein 1 (EtMIC1, accession number AAD03350), Neospora caninum microneme protein 2 (NcMIC2, accession number AAF01565), and Cryptosporidium parvum thrombospondin-related anonymous protein-C1 (CpTRAP-C1, accession number AAC48311). Molecular Cell 2003 11, 885-894DOI: (10.1016/S1097-2765(03)00113-8)

Figure 2 The MIC2 C Domain Binds Specifically to a Complex of Parasite Proteins (A) A complex of five major bands (designated 1–5) found in parasite lysates was specifically bound by a GST fusion to the MIC2 C domain (GST-MIC2t). Extracts from T. gondii were incubated with GST or GST-MIC2t and specifically bound proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining. The parasite lysate (Tg Lysate) shown in the second lane represents 1% of the material employed in the + lysate pull-down lanes. * indicates the position of GST-MIC2t; ** indicates the position of GST. Molecular mass in kilodaltons (kDa); LMW refers to low-molecular weight markers. (B) While aldolase and actin were part of the complex of proteins specifically pulled down by GST-MIC2t, parasite myosin (TgMyo-A) was not. Samples identical to the lanes shown in (A) were subjected to immunoblotting with an aldolase (α ALD)-, actin (α ACT)-, and myosin-A (α MYO)-specific antibodies (shown to the right of blots). Molecular mass is in kilodaltons. (C) MIC2 coimmunoprecipitated parasite aldolase and actin from parasite cell lysates. MIC2 was immunoprecipitated (IP) using rat anti-MIC2, resolved by SDS-PAGE, and immunoblotted with MIC2 (α MIC2)-, aldolase (α ALD)-, or actin (α ACT)-specific antibodies. Antiserum from the preimmunized rat (Prebleed) was used as the control. * indicates the position of the aldolase protein in the anti-MIC2 lane. Heavy (HC) and light (LC) chains of the rat immunoglobulins employed in the pull down were recognized by the anti-aldolase antibody. Molecular mass is in kilodaltons. Rabbit aldolase was included in lane 1, and total parasite lysate (Tg lysate) was included in lane 2 as positive controls. (D) Parasite aldolase binds directly to actin. Toxoplasma aldolase fused to GST (GST-TgALD) or GST alone were incubated in buffer A, ±25 μg of rabbit actin (Rb Actin). Proteins bound to glutathione beads were eluted and resolved by SDS PAGE. Top gel is Coomasie blue stained. Bottom gel is a Western blot with mAb C4 that is specific for actin (α ACT). LWM refers to low molecular weight markers. Molecular Cell 2003 11, 885-894DOI: (10.1016/S1097-2765(03)00113-8)

Figure 3 Aldolase Associates with MIC2 and TRAP in a Tryptophan-Dependent Manner (A) The tryptophan residue of the C domain of MIC2 mediates the specific association with parasite aldolase and actin. Extracts from T. gondii were incubated with GST fused to the MIC2 C domain (GST-MIC2t) or to a GST-MIC2-C domain mutant where tryptophan was replaced with alanine (GST-MIC2tW/A). Specifically bound proteins (designated as bands 1–5) were resolved by SDS-PAGE, visualized by Coomassie blue staining. (B) Similar samples to those shown in (A) were transferred to nitrocellulose and immunoblotted with antibodies to aldolase (α ALD) (top panel) or actin (α ACT) (bottom panel). The position of the aldolase positive band in the top panel corresponds to band 1 (indicated by 1). Molecular mass is in kilodaltons. (C) Aldolase was specifically pulled down by GST fusions to the C domains of MIC2 (GST-MIC2t) or TRAP (GST-TRAPt). This interaction was disrupted by tryptophan to alanine mutations in the C domains of MIC2 or TRAP (GST-MIC2tW/A and GST-TRAPtW/A, respectively). Purified rabbit aldolase (RAld) (added in lane 1 as a control) was incubated with GST or fusion proteins, and specifically bound proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining. Molecular mass is in kilodaltons. (D) Purified recombinant Toxoplasma aldolase-His6 (TgAld) also bound specifically to GST-MIC2t but not the W/A mutant. SDS PAGE gel was visualized by Coomasie blue staining. Molecular mass is in kilodaltons. Molecular Cell 2003 11, 885-894DOI: (10.1016/S1097-2765(03)00113-8)

Figure 4 Parasite Aldolase Is Apically Localized but Not Secreted (A) Aldolase was concentrated at the anterior end of the parasite where it largely colocalized with the secretory protein MIC2 (top panel). In contrast, pyruvate kinase (PK) was uniformly distributed in the cytoplasm (bottom panel). Top panel: aldolase was visualized using goat anti-aldolase and donkey anti-goat conjugated to Alexa 488. MIC2 was localized using rabbit anti-MIC2 and donkey anti-rabbit conjugated to Alexa 594. Bottom panel: PK was localized using rabbit anti-PK and goat anti-rabbit conjugated to Alexa 488. MIC2 was localized using mouse anti-MIC2 and goat anti-mouse conjugated to Alexa 594. Scale bars, 5 μm. (B) Aldolase was not secreted, unlike MIC2 and M2AP. Freshly harvested tachyzoites were stimulated to undergo protein secretion and the intact parasites (Cell pellet) or culture supernatants (Secreted) were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and subjected to immunoblotting with MIC2 (α MIC2)-, M2AP (α M2AP)-, or aldolase (α ALD)-specific antibodies. Molecular mass is in kilodaltons. Rabbit aldolase (100 ng) was added in lane 1 as a positive control. Molecular Cell 2003 11, 885-894DOI: (10.1016/S1097-2765(03)00113-8)

Figure 5 Aldolase Redistributes in Association with MIC2 in Gliding Parasites (A) Motile parasites deposited MIC2 in trails formed on the substrate. Parasites were allowed to glide on poly-L-lysine-coated glass, fixed, permeabilized, and stained for MIC2 using mAb 6D10 followed by donkey anti-mouse-IgG conjugated to Alexa 594. MIC2 is observed in the anterior end of the parasite (within micronemes), dispersed in small clusters along the parasite surface, concentrated at the posterior end, and deposited in trails formed on the substrate. Scale bar, 5 μm. (B) In parasites with extruded conoids (arrow), both MIC2 and aldolase were redistributed to the apical tip of the parasite. MIC2 was revealed by mAb 6D10 followed by donkey anti-mouse-IgG conjugated to Alexa 594. Aldolase was revealed by goat-anti-aldolase followed by donkey anti-goat-IgG conjugated to Alexa 488. Scale bar, 5 μm. (C) Enlargement of the image shown in (A), revealing that in parasites where MIC2 was localized at the posterior end of the cell; aldolase was also partially redistributed to this region (arrow). IF staining is as in (B). Scale bar, 5 μm. Molecular Cell 2003 11, 885-894DOI: (10.1016/S1097-2765(03)00113-8)

Figure 6 Model for Aldolase-Linked Motility in Apicomplexan Parasites During gliding motility, the cell surface adhesin MIC2 is translocated toward the posterior end of the cell in an actin-dependent process. The motor protein TgMyoA is anchored on the inner membrane complex (IMC). Myosin interacts with actin filaments in a nonprocessive manner, walking toward the plus (barbed) end via its actin-dependent ATPase activity. Aldolase, depicted as a tetramer, provides a bridge between filamentous actin and the cytoplasmic domain of MIC2. Aldolase binding depends on a single tryptophan residue in the C domain of MIC2. Backward translocation of the adhesin, bound to its cognate receptor on the host cell surface or substrate, is responsible for driving the parasite forward. Molecular Cell 2003 11, 885-894DOI: (10.1016/S1097-2765(03)00113-8)