| 2001 |
TgM2AP physically associates with TgMIC2, forming a complex within 15 min of synthesis. M2AP remains associated with MIC2 in the micronemes, on the parasite surface during invasion, and in the culture medium after release. M2AP is proteolytically processed: its propeptide is removed during transit through the Golgi, and a second cleavage occurs on the parasite surface after discharge from micronemes. |
Co-immunoprecipitation, pulse-chase labeling, immunofluorescence, immunoelectron microscopy |
Molecular microbiology |
High |
11532123
|
| 2003 |
Disruption of M2AP (knockout) caused >80% impairment in host cell entry. MIC2 partially accumulated in the ER and Golgi in M2AP-KO parasites, and rapid secretion of MIC2 upon host cell attachment was abolished, demonstrating that M2AP is required for correct trafficking and secretion of MIC2. |
Genetic knockout, immunofluorescence, invasion assays, secretion assays |
The EMBO journal |
High |
12727875
|
| 2003 |
MIC2 and M2AP form a stable hexameric complex (~450 kDa) consisting of three alpha-beta dimers, as determined by native blue gels and gel filtration. The cytoplasmic C-domain of MIC2 is not required for protein trafficking or secretion, and mutant MIC2 lacking this domain is escorted to micronemes by association with endogenous wild-type MIC2. |
Native PAGE (blue native gels), gel filtration chromatography, transgenic parasite expression |
The Journal of biological chemistry |
High |
14670959
|
| 2006 |
Conditional suppression of MIC2 caused mistrafficking of M2AP, demonstrating mutual dependence: MIC2 is required for correct M2AP sorting to the micronemes, and M2AP is required for efficient MIC2 trafficking and secretion. MIC2-deficient parasites showed markedly defective host-cell attachment, loss of helical gliding motility, and were unable to establish lethal infection in mice. |
Conditional (anhydrotetracycline-regulated) expression system, knockout, immunofluorescence, invasion/attachment assays, murine infection model |
PLoS pathogens |
High |
16933991
|
| 2006 |
The M2AP propeptide and its proteolytic removal influence distinct aspects of MIC2-M2AP trafficking and function. ProM2AP traffics through endosomal compartments; deletion of the propeptide leads to defective trafficking near this site causing aberrant MIC2 processing and decreased secretion. Mutation of cleavage-site residues allows normal localization but reduces complex stability and secretion from micronemes. Both manipulations impair invasion and reduce virulence in vivo. |
Propeptide deletion and cleavage-site mutagenesis, immunofluorescence, invasion assays, in vivo murine virulence model |
Molecular biology of the cell |
High |
16914527
|
| 2014 |
The NMR solution structure of the globular domain of M2AP was solved; M2AP adopts a modified galectin fold similar to the C-terminal domain of MIC1. NMR and immunoprecipitation analyses showed that hydrophobic residues on one face of the M2AP galectin fold bind to the membrane-proximal sixth thrombospondin type I repeat domain (TSR6) of MIC2. |
NMR structure determination, immunoprecipitation |
The Journal of biological chemistry |
High |
25411252
|
| 2014 |
Crystal structures of proMIC2 and MIC2 N-terminal fragments (VWA + TSR1 domains) were solved. The MIC2-M2AP ectodomain complex is a highly elongated 1:1 monomer with M2AP bound to the TSR6 domain. The MIC2 VWA domain adopts a closed conformation in the crystal structure, and TSR1 contains a proline-rich disulfide-bonded pigtail loop overlapping the VWA domain. |
X-ray crystallography, biochemical binding assays |
Proceedings of the National Academy of Sciences of the United States of America |
High |
24639528
|
| 2023 |
Crystal structures of M2AP alone and in complex with TSR6 of MIC2 were solved. TSR6 residue H620 is the hotspot at the center of the binding interface with M2AP. Residue Y602 of TSR6 is dynamic, equilibrating between layered and flipped-out states; M2AP binding shifts Y602 to the flipped-out state. The non-layered side of TSR6 mediates binding to M2AP. |
X-ray crystallography of M2AP alone and M2AP-TSR6 complex |
Communications biology |
High |
37652989
|
| 2011 |
Forward targeting of M2AP to micronemes requires a valine residue at position +3 relative to signal peptidase cleavage in the propeptide. Mutagenesis of this valine abolished correct micronemal trafficking. Propeptide domains are functionally interchangeable between T. gondii MICs and with Eimeria tenella MIC5 propeptide, indicating a conserved mechanism. |
N-terminal deletion and point mutagenesis of propeptide, immunofluorescence-based trafficking assay |
Traffic (Copenhagen, Denmark) |
High |
21438967
|
| 2008 |
The M2AP propeptide functions as a forward-targeting element for micronemal trafficking. When the M2AP propeptide was exchanged for the MIC5 propeptide, correct trafficking was maintained, indicating propeptides are functionally interchangeable between microneme proteins. |
Propeptide exchange constructs, immunofluorescence localization |
Biology of the cell |
Medium |
17995454
|
| 2005 |
The M2AP propeptide is required for the MIC2-M2AP complex to exit from the early endosome. A cleavage-resistant M2AP mutant reached micronemes but could not be rapidly mobilized to the parasite surface. Both the propeptide deletion and cleavage-resistant mutants were unable to support normal parasite invasion and were partially attenuated in virulence. MIC2 was also shown to be required for correct M2AP sorting to micronemes. |
M2AP propeptide deletion and cleavage-resistant mutant expression in M2AP-KO background, conditional MIC2 expression, immunofluorescence, invasion assays, murine virulence model |
Parasitology research |
High |
16385407
|
| 2004 |
An M2AP deletion mutant (m2ap KO) was used to show that the MIC2 adhesin specifically contributes to calmidazolium-stimulated gliding motility, implicating the MIC2-M2AP complex in calcium-regulated gliding. |
Deletion mutant phenotypic analysis, gliding motility assay with calmidazolium stimulation |
Journal of cell science |
Medium |
15507483
|
| 2004 |
In the absence of TgMIC2, TgM2AP was incorrectly processed and mistargeted to the parasitophorous vacuole instead of the micronemes, demonstrating that MIC2 is required for correct M2AP maturation and trafficking. EtMIC1 (Eimeria tenella ortholog of MIC2) could substitute for TgMIC2 to allow parasite survival but could not fully restore M2AP trafficking or invasion efficiency. |
Trans-genera complementation (EtMIC1/EtMIC2 introduced into T. gondii), TgMIC2 knockout, immunofluorescence, invasion assays |
Cellular microbiology |
High |
15236644
|
| 2010 |
TgSUB1 (a GPI-anchored microneme subtilisin protease) is responsible for proteolytic trimming of M2AP on the parasite surface after secretion. Parasites lacking TgSUB1 fail to process M2AP (and MIC2, MIC4) after surface release, leading to decreased cell attachment, reduced gliding, and lower invasion rates. |
TgSUB1 knockout and complementation (including GPI-anchor deletion mutant), secretion/processing assays, invasion and attachment assays |
Cellular microbiology |
High |
20678172
|