Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal 6HA-tagged version of ISP1
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
To confirm the polarized localization of ISP1 and ISP3 in the zygote, we tagged individual endogenous ISP1 or ISP3 (RMgm-4894) with a sextuple HA epitope (6HA) at the C-terminus, obtaining the isp1::6HA and isp3::6HA parasites. Both proteins were expressed in asexual blood stages, gametocytes, ookinetes, and oocysts. Time-course expression analysis showed that both proteins were 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, in agreement with previous observations in P. berghei. Mature ookinetes (stage V) still had peripheral distribution of both ISP1 and ISP3 proteins in whole cell. We further generated a doubly tagged parasite (DTS), isp1::3V5/isp3::6HA (RMgm-4895), by tagging endogenous ISP1 with a triple V5 epitope (3V5) in the isp3::6HA parasite and observed similar co-localization of ISP1/ISP3 at the periphery of zygote protrusion. The localization patterns of ISP1/ISP3 further 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.
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