A transient forward-targeting element for microneme-regulated secretion in Toxoplasma gondii

Abstract

Background information: Accurate sorting of proteins to the three types of secretory granules in Toxoplasma gondii is crucial for successful cell invasion by this obligate intracellular parasite. As in other eukaryotic systems, propeptide sequences are a common yet poorly understood feature of proteins destined for regulated secretion, which for Toxoplasma occurs through two distinct invasion organelles, rhoptries and micronemes. Microneme discharge during parasite apical attachment plays a pivotal role in cell invasion by delivering adhesive proteins for host receptor engagement.

Results: We show here that the small micronemal proprotein MIC5 (microneme protein-5) undergoes proteolytic maturation at a site beyond the Golgi, and only the processed form of MIC5 is secreted via the micronemes. Proper cleavage of the MIC5 propeptide relies on an arginine residue in the P1' position, although P1' mutants are still cleaved to a lesser extent at an alternative site downstream of the primary site. Nonetheless, this aberrantly cleaved species still correctly traffics to the micronemes, indicating that correct cleavage is not necessary for micronemal targeting. In contrast, a deletion mutant lacking the propeptide was retained within the secretory system, principally in the ER (endoplasmic reticulum). The MIC5 propeptide also supported correct trafficking when exchanged for the M2AP propeptide, which was recently shown to also be required for micronemal trafficking of the TgMIC2 (T. gondii MIC2)-M2AP complex [Harper, Huynh, Coppens, Parussini, Moreno and Carruthers (2006) Mol. Biol. Cell 17, 4551-4563].

Conclusion: Our results illuminate common and unique features of micronemal propeptides in their role as trafficking facilitators.

Figure 1

proMIC5 occupies a post-Golgi compartment(s) and DG. (A) Immunoblot of parasite lysate showing that αMIC5 antibodies recognize both proMIC5 and mature MIC5 whereasαMIC5propeptide antibodies recognize only proMIC5. (B) IFA of extracellular RH parasites stained with αMIC5propeptide and αMIC2 (panel Ba), GRASP52-mRFP (GRASP; panel Bb), GRA4 (panel Bc) or proM2AP (panel Bd). Arrow denotes staining in a post-Golgi compartment(s). Scale: 5 μm. (C) Dual labelling immunoelectron microscopy withαMIC5propeptide (10 nm gold) and αMIC5 (5 nm gold) antibodies. Rectangular box indicates region expanded in C′. Arrows denote intensely stained vesicular structures that are mostly, although not exclusively, in the post-Golgi region. DG, dense granule; Go, Golgi; M, micronemes; N, nucleus; R, rhoptry. (D) A subset of DG was recognized by both αMIC5propeptide (10 nm gold) and to a lesser extent by αMIC5 (5 nm gold). Scale for C, C′, and D: 200nm.

Figure 2

ProMIC5 and mature MIC5 are secreted via different pathways. Immunoblot of excreted/secreted antigen from RH parasites treated with (+) or without (−) B-AM, probed with αMIC2 (positive control), αGRA1 (negative control), or αMIC5 antibodies.

Figure 3

P1′ residue mutants are aberrantly processed but show normal trafficking to micronemes. (A) Schematic of propeptide cleavage site mutants used. The wild-type MIC5 (M5) sequence is shown at the top and an arrow depicts the normal cleavage site. Mutated residues in myc-tagged mutants are delineated by black boxes. (B) Immunoblots of propeptide cleavage site mutants probed with αMIC5 and αactin (loading control). MIC5-myc serves as a myc-tagged, wild type control. The pro form of MIC5P1P1’-myc is larger because of an inadvertent additional mutation at the P3′ position. Mutation at the P1′ site diminishes processing of proMIC5-myc to MIC5-myc. (C) N-terminal microsequencing of affinity purified MIC5P1’-myc reveals cleavage at a site 6 amino acids downstream of the wild-type cleavage site. “(A)” indicates the alanine substitution in the P1′ position of MIC5P1’-myc. Underlined are residues identified in the first 5 cycles of Edman degradation. (D) IFA of extracellular MIC5-myc and MIC5P1’-myc parasites stained with αMIC5 and αAMA1, showing extensive localization of MIC5P1’-myc in the micronemes. (E) IFA with αMIC5 and αGRA1 indicates little or no diversion of MIC5P1’-myc to DG. Scale for D and E: 10 μm.

Figure 4

The MIC5 propeptide is necessary for microneme targeting. (A) Schematic of myc-tagged mutants showing native MIC5, wild type MIC5-myc, and MIC5Δpro-myc. (B) Immunoblots of parasite lysates probed with αMIC5 showing the absence of proMIC5 in MIC5Δpromyc parasites. Actin is included as a loading control. (C) IFA of extracellular (upper panels) or intracellular (lower panels) MIC5-myc or MIC5Δpro-myc parasites stained withαMIC5 and αAMA1. Note the wide distribution of MIC5Δpro-myc throughout the cytoplasm of extracellular parasites and the perinuclear, ER staining in intracellular parasites. Scale: 5 μm. (D) Immunoblots of ESA fractions treated with (+) and without (−) B-AM show that MIC5Δpro -myc is secreted via a non-micronemal pathway.

Figure 5

The MIC5 propeptide supports normal stability, secretion, and trafficking of the MIC2-M2AP complex. (A) Immunoblot of parasite lysates probed with αMIC2 and αM2AP showing minimal MIC2 degradation (MIC2deg) in parasites expressing the MIC5 propeptide fused to M2AP (M5proM2AP). An asterisk denotes an additional putative degradation product of M5proM2AP. (B) Quantification of MIC2 degradation by direct detection of chemiluminescent emissions from immunoblots. Values are expressed as the average ratio of MIC2 to MIC2deg from 3 independent experiments. (C) Immunoblot of ESA fractions collected from ethanol- stimulated parasites and probed with αMIC2 and αAMA1 (loading control). Note the normal secretion of MIC2 from M5proM2AP parasites in contrast to weak secretion from ΔproM2AP and M2APKO parasites. (D) IFA localization of M2AP in M5proM2AP parasites (middle panels) shows normal distribution in the micronemes (counterstained for AMA1), similar to 1C4 (upper panels), but in contrast to secretory retention (arrow) or release into the parasitophorous vacuole (arrowhead) of M2AP in ΔproM2AP parasites (lower panels). Scale bar: 5 μm.