Mutant/mutation
The mutant expresses a mutated form of IMC1g: a fusion of IMC1g with HA-tagged mutant E. coli DHFR (cDDHA). cDD binds trimethoprim (TMP) and is stable, but undergoes proteasomal degradation in the absence of trimethoprim

Protein (function)
The inner membrane complex (IMC), a double-membrane organelle underneath the plasma membrane in apicomplexan parasites, plays a significant role in motility and invasion and confers shape to the cell. On the cytosolic side of the IMC, it is associated tightly with the subpellicular protein network (SPN), which supports the IMC and provides tensile strength to the parasite. Besides the critical role of IMC in regulating gliding motility, it also confers stability to cellular architecture and provides a scaffolding framework in parasite division
Alveolins (or IMC1 proteins) are major SPN components, broadly conserved throughout the alveolate and important for IMC-SPN interconnection
The imc1h gene belongs to small 'family' of conserved genes that express putative membrane skeleton proteins which contain domains that share sequence homology to domains of articulins, proteins of membrane skeleton of free-living protists and show homology to the inner membrane complex protein 1 (TgIMC1) of the subpellicular network of Toxoplasma gondii tachyzoites. In Plasmodium spp., 13 conserved IMC1 family members, namely, IMC1a-m, have been identified.
PbIMC1g (PbANKA_1240600) is predicted to encode a ~34.3 kDa protein lacking transmembrane domains or a signal peptide. PbIMC1g is a palmitoylated protein.
IMC1g orthologs in Plasmodium spp. share a conserved IMCp domain (Pfam 12314) and harbors a core sub-repeat motif “EKI(V)V(I)EVP” within the IMCp domain that defines the alveolins

Phenotype
Multiple attempts to knock out pbimc1g has failed, indicating that it is essential for asexual parasite replication. Therefore, we generated the 'conditional knockdown (KD)' PbIMC1g(cKD) parasite (see RMgm-5635) by tagging the C-terminus of the endogenous pbimc1g gene with a triple HA tag and a dihydrofolate reductase (DHFR)-based destabilizing domain (3×HA-DDD) for conditional KD of PbIMC1g using the DDD system. This parasite expresses a PbIMC1g-HA-DDD fusion protein.
By treating parasites and mice with Trimethoprim (TMP), evidence is presented that
- Knock down of IMC1g expression results in incomplete cytokinesis and formation of IMC and thus that that PbIMC1g is important for asexual stage schizogony.
- PbIMC1g is required for male gametogenesis 
Compared to the [+] TMP group, [−] TMP did not affect gametocytemia or the male-to-female sex ratio. Moreover, the proportions of macrogametocyte activation and macrogamete formation were comparable between the [+] and [−] TMP parasites. However, the number of exflagellation centers in [−] TMP parasites was significantly reduced compared to [+] TMP parasites
- Knock down of IMC1g expression results in impaired ookinete conversion
-  PbIMC1g is not necessary for subpellicular microtubule organization in developing ookinetes
- Knock down of IMC1g expression affects ookinete motility and mosquito transmission

Additional information
See mutant RMgm-5634 expressinga C-terminal 3xHA tagged version of IMC1g. Analysis of this mutant showed expression in blood stages, gametocytes and ookinetes

PbIMC1g has an IMCp domain (aa 29–184) and a C-terminal region (aa 185–297). To assess which domain is needed for proper protein localization, we generated deletion constructs of PbIMC1g for episomal expression in wild-type P. berghei. Episomal expression of mCherry-tagged full-length PbIMC1g protein (FL) showed a similar localization pattern as PbIMC1g-HA. C-terminal deletion (ΔC) also localized the protein to the merozoite periphery, albeit diffused signals in the parasite cytoplasm were also visible. In contrast, deletion of the IMCp domain (ΔIMCp) completely abolished the peripheral localization pattern of the protein, indicating that he IMCp domain and, to a lesser extent, the C-terminal region of PbIMC1g protein are needed for its efficient IMC association. 

Protein palmitoylation, catalyzed by aspartate–histidine–histidine–cysteine (DHHC) palmitoyl acyltransferases in Plasmodium, plays a vital role in targeting proteins to the IMC and mediating protein-protein interactions. PbIMC1g has seven predicted palmitoylation sites at Cys residues 2, 10, 11, 233, 238, 255, and 296. To evaluate the importance of the predicted PbIMC1g palmitoylation sites in proper IMC targeting, we generated two PbIMC1g expression constructs in which the cysteine residues at N-terminus (C2, C10, and C11) and C-terminus (C233, C238, C255, and C296) were mutated to alanine, respectively. Disruption of the palmitoylation sites with both constructs resulted in noticeable mis-localization of the protein to the parasite cytoplasm in schizonts. However, the localization pattern of Palm-mutN and Palm-mutC at the ookinete stage was not altered. 
Compared to the palmitoylation prediction core of the C-terminus, the N-terminal of PbIMC1g contains higher predicted scores of palmitoylation sites. To better understand how palmitoylation regulates PbIMC1g function, we generated a transgenic strain (Nmut) with N- (C2, C10, and C11) palmitoylation sites mutated to alanines using CRISPR-cas9 genome editing technology. The Nmut parasites proliferated, with a parasitemia of ~63.4% at day 17 post-infection (dpi), comparable to WT P. berghei. Meanwhile, the Nmut strain-infected group also exhibited a comparable survival span to the WT group, indicating that N-terminal palmitoylation of PbIMC1g is not required for its function in asexual-stage proliferation. However, the Nmut strain exhibited a significant 100%, 44.8%, and 100% reduction in male and female gametocytemia and male/female gametocyte ratio at 3 dpi. No male gametocytes and only a few unmatured female gametocytes were observed in the Nmut strain at 3 dpi. Furthermore, neither exflagellation center formation nor ookinete formation was observed in the Nmut strain at 3 dpi. Cross-fertilization assay using parasite lines defective in either female (Δp47) or male gametes (Δp48/45) revealed that both male and female gametes’ function was impaired in Nmut parasites. Therefore, we conclude that the palmitoylation sites are necessary for incorporating PbIMC1g into the IMC in the schizont stage and are essential for parasites’ gametocytogenesis.

Multiple attempts to knock out pbimc1g has failed, indicating that it is essential for asexual parasite replication. Therefore, we generated the 'conditional knockdown (KD)' PbIMC1g(cKD) parasite (see RMgm-5635) by tagging the C-terminus of the endogenous pbimc1g gene with a triple HA tag and a dihydrofolate reductase (DHFR)-based destabilizing domain (3×HA-DDD) for conditional KD of PbIMC1g using the DDD system. This parasite expresses a PbIMC1g-HA-DDD fusion protein.
By treating parasites and mice with Trimethoprim (TMP), evidence is presented that
- Knock down of IMC1g expression results in incomplete cytokinesis and formation of IMC and thus that that PbIMC1g is important for asexual stage schizogony.
- PbIMC1g is required for male gametogenesis 
Compared to the [+] TMP group, [−] TMP did not affect gametocytemia or the male-to-female sex ratio. Moreover, the proportions of macrogametocyte activation and macrogamete formation were comparable between the [+] and [−] TMP parasites. However, the number of exflagellation centers in [−] TMP parasites was significantly reduced compared to [+] TMP parasites
- Knock down of IMC1g expression results in impaired ookinete conversion
-  PbIMC1g is not necessary for subpellicular microtubule organization in developing ookinetes
- Knock down of IMC1g expression affects ookinete motility and mosquito transmission

Evidence is presented that the P. vivax imc1g ortholog (PVX_079955) PvIMC1g is functionally equivalent to PbIMC1g in the transgenic P. berghei.
We replaced the pbimc1g coding sequence with P. vivax imc1g ortholog (PVX_079955), while simultaneously tagging the PvIMC1g with a 2×Myc tag. Successful allele replacement was confirmed by diagnostic PCR, and the PvIMC1g-Myc protein expression in a transgenic P. berghei parasite line (PvIMC1gTR) was confirmed by western blotting using the anti-Myc mAb. The PvIMC1g-Myc protein exhibited a similar localization profile as in the PbIMC1gHA transgenic parasite

Other mutants