Additional remarks phenotype | Mutant/mutation
The mutant expresses a mutated ISP1 with N-terminal cysteines (C7/C8) replaced with A7/A8 (alanine). in addition ISP is tagged with the BFP::3V5 reporter
Protein (function)
The inner membrane complex (IMC)-residing protein ISP (IMC subcompartment protein) is restricted to the phylum apicomplexa and plays key roles in parasite biology. Two ISP members, ISP1 and ISP3, are found in Plasmodium parasites, and display clear apical polarity of expression in zygotes, suggesting possible role in zygote protrusion or elongation. However, parasites with disruption of ISP1 display a modest decrease in zygote-to-ookinete differentiation, while ISP3 depletion has no significant effect on this process.
Phenotype
Both ISP1 and ISP3 have two N-terminal cysteines (C7/C8 in ISP1 and C6/C7 in ISP3) that can potentially be palmitoylated for IMC targeting as shown in schizonts of P. falciparum . Using resin-assisted capture (Acyl-RAC) method, we detected palmitoylation of ISP1 and ISP3 in the zygotes. To study the effect of C7/C8 palmitoylation on protein IMC localization, we generated an ISP1 mutant by replacing C7/C8 with A7/A8 (alanine) and fused the mutant ISP1 with BFP::3V5 reporter. The C7A/C8A mutant protein lost palmitoylation and was localized in the cytoplasm when episomally expressed in WT parasites. In addition, glycine-to-alanine substitution at position 2 (G2A) abolished ISP1 palmitoylation and IMC localization, supporting the observations that glycine myristoylation enhances palmitoylation of the adjacent cysteine.
In wild type zygotes ookinetes ISP1 and ISP3 are concentrated at an apical dot in zygotes (stage I), with residual ISP3 distributed at cytoplasm. In stage III, both proteins are expressed at the periphery of the protrusion, but not zygote remnant. These results support that ISP1/ISP3 is localized in IMC, but not PPM during this differentiation. The localization patterns of ISP1/ISP3 suggest critical roles of these two proteins in zygote protrusion and/or elongation.
Additional information
From the Abstract:
'Here, we show that palmitoylation of N-terminal cysteines of two inner membrane complex (IMC) proteins (ISP1/ISP3) regulates the IMC localization of ISP1/ISP3 and zygote-to-ookinete differentiation. Palmitoylation of ISP1/ISP3 is catalyzed by an the IMC-residing palmitoyl-S-acyl-transferase (PAT) DHHC2. IMC-anchored ISP1 and ISP3 interact with microtubule component b-tubulin, serving as tethers to maintain the proper structure of subpellicular microtubules (SPMs) during zygote elongation.
Other mutants |