Fertilization and ookinete | Compared to WT DHHC2, the catalytic-deficient DHHC2C128A failed to restore zygote polarization of ISP1 as well as ookinete differentiation of the dhhc2kd (RMgm-4898), indicating that the PAT activity is essential for DHHC2 function. Surprisingly, this C128A mutation in the DHHC motif led to cytoplasmic distribution of DHHC2, indicating that its own PAT activity is also required for the proper localization of DHHC2. |
Additional remarks phenotype | Mutant/mutation
The mutant expresses a mutated form of DHHC2 (glycine 128 changed to alanine). In addition, line expresses C-terminal tagged forms of ISP1 (6xHA tag)
Protein (function)
Many proteins are post-translationally modified by the addition of lipids. Palmitoylation results in the addition of a C-16 fatty acid to a cysteine residue within a given protein. Palmitoylation is reversible and thus can dynamically regulate a protein’s subcellular localization, gene expression and activity. There are 11 predicted palmitoyl-S-acyl-transferases (PATs; named DHHC1–11) in the genomes of P. yoelii and P. berghei parasites.
Phenotype
Replacing cysteine to alanine within DHHC motif impairs the catalytic activity of PATs. Compared to WT DHHC2, the catalytic-deficient DHHC2C128A failed to restore zygote polarization of ISP1 as well as ookinete differentiation of the dhhc2kd (RMgm-4898), indicating that the PAT activity is essential for DHHC2 function. Surprisingly, this C128A mutation in the DHHC motif led to cytoplasmic distribution of DHHC2, indicating that its own PAT activity is also required for the proper localization of DHHC2. Together, these results demonstrate that the palmitoylation of ISP1/ISP3 by DHHC2 mediates their IMC targeting, which is essential for zygote-to-ookinete differentiation.
Additional information
DHHC2 possesses a 75 aa (residue 210–284) cytosolic C-terminus conserved among Plasmodium spp. Hydrophilicity analysis revealed high hydrophobicity in a segment of residue 251–261. To test whether this C-terminus regulates DHHC2 localization at zygote, we episomally expressed three 6HA-tagged DHHC2, each with a truncated C- terminus of different lengths (ΔC1: missing residues 210–232, ΔC2: missing 233–262, and ΔC3 missing: 263–284). The expression of all three mutants was comparable to that of WT in zygotes. However, only ΔC2 lost IMC targeting and polarized expression, suggesting that the C2 segment is required for proper DHHC2 trafficking. Sequence alignment revealed four conserved cysteine residues (C255, C258, C260, and C262) in C2 segment, and palmitoylation of these cysteines likely plays a role in IMC targeting of DHHC2. Replacement of all four cysteine with alanine (C255A/C258A/C260A/C262A:C4A) completely abolished the palmitoylation of DHHC2, leading to cytoplasmic distribution of the DHHC2 in zygotes and retorts.
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 |