Mutant/mutation
The mutant expresses a mutated form of akratin that is C-terminally GFP-tagged. In the mutated akratin the C-terminal EYKK motif has been changed into four alanines and a GFP tag is added (C-terminal). The mutated and tagged akratin gene has been introduced into the akratin locus of a mutant (see RMgm-4885) in which the akratin gene has been deleted.
Published in bioRxiv preprint doi: https://doi.org/10.1101/2020.09.29.318857.

Protein (function)
PbANKA_1105300 was found to be conserved among Plasmodium spp. It shares 82% identity with its orthologue in P. yoelii, but only 35% and 32% with its orthologues in P. falciparum and P. vivax, respectively. In contrast to P. berghei and P. falciparum, the orthologues in P. yoelii and P. vivax are predicted to contain only two TMDs. Furthermore, the P. falciparum protein contains about two times more amino acids than the P. berghei protein. No orthologue was found outside Plasmodium spp

Phenotype
Mutation of a C-terminal motif of akratin uncoupled akratin function in gametocytes and ookinetes (see mutant RMgm-4885). In the mutant expressing mutated akratin, ookinetes were formed but no oocysts. Evidence is presented that the mutant ookinetes are defective in midgut-wall traversal.
The mutant lacking expression of akratin (see mutant RMgm-4885) fails to produce male gametes (and ookinetes)

Additional information
In mammalian cells the C-terminal sorting sequence YXXf (with Y, tyrosine; X, any amino acid and f, hydrophobic amino acid) is important for protein trafficking. A similar motif was also shown to be important for trafficking of the micronemal sporozoite protein TRAP (Bhanot et al., 2003), although the sequence is annotated as being located in the trans-membrane domain. Somewhat similar tyrosine-containing C- terminal motifs (SYHYY and EIEYE) from the Toxoplasma gondii adhesin MIC2 were also shown to target the protein to micronemes. We noticed in the C-terminal, likely cytoplasmic domain of P. berghei akratin a short sequence (YKKL) representing the described YXXf pattern. However, since the P. falciparum orthologue contains a leucine instead of a tyrosine and a conserved glutamic acid to the left we chose to mutate the sequence EYKK instead of YKKL to investigate a possible role for akratin trafficking. To this end, we generated a mutant parasite line expressing akratin-GFP with the EYKK motif changed into four alanines.

The Akratin-EYKK/AAAA-GFP mutant showed similar staining patterns in asexual and sexual parasite stages compared with akratin-GFP. (see mutant RMgm-4888) However, in contrast to the mostly peripheral and vesicular localization of akratin-GFP in ookinetes the akratin-EYKK/AAAA GFP signal was diffusely localized within the cytoplasm

Other mutants