Mutant/mutation
The mutant expresses a C-terminal TurboID::GFP-tagged version of ALBA4

Protein (function)
ALBA proteins are evolutionarily conserved from Archaea and consistently have been found to bind to nucleic acids. Structurally, the ALBA domain resembles the IF3 C-terminal domain, which binds to the small subunit of the prokaryotic ribosome. Together, this strategy provides a straightforward mechanism for ALBA proteins to tether nucleic acids to the ribosome. Plasmodium spp. contain at least four ALBA-domain containing proteins, and have been shown to bind both DNA and RNA. Of these ALBA proteins, ALBA4 is specific to the Apicomplexan lineage and its Chromerid ancestor (40-50%; identity / 60-65% similarity).

Phenotype
In order to determine mechanisms that may control translation across host-to-vector transmission, we assessed the composition of the DOZI/CITH/ALBA (DCA) translationally repressive protein complex before and after transmission. We and others have identified components of the DCA complex in P. yoelii and P. berghei via IP/MS in asexual blood stages, gametocytes, and oocyst sporozoites either with or without crosslinking. However, it was not clear how the DCA complex may respond to transmission cues that are predicted to cause the release of its bound and regulated mRNAs and thus relieve translational control. Therefore, we employed proximity proteomics by fusing a variant of E. coli biotin ligase and GFPmut2 to the C-terminus of DOZI and ALBA4 as experimental bait proteins, or as an unfused control to account for highly abundant proteins in the same compartment (i.e., cytosol). We chose this approach to not only determine the protein composition of the DCA complex across host-to-vector transmission, but also to determine if spatial organization of this complex changes. We selected DOZI and ALBA4 as they are known to associate with the 5’ or 3’ end of bound mRNAs, respectively, and thus provide protein proxies for the two ends of target mRNAs.
We initially tested the first generation BioID enzyme before adopting the use of the further engineered TurboID (TID) ligase that allowed for more robust labeling in a shorter labeling time, as we and others have recently shown for P. falciparum and P. berghei.To establish an appropriate duration of labeling, we assessed in vivo biotinylation with supplementation with 150 μM biotin for increasing time, then used total parasite lysate for affinity blots probed with streptavidin-HRP for both bait parasite lines, ALBA4::TurboID::GFP and DOZI::TurboID::GFP. We found that one hour of biotin supplementation was sufficient for robust biotin labeling by TurboID in total parasite lysates for both female gametocytes and zygotes. Therefore, we used these labeling conditions for all experiments.
Biotinylated proteins were able to be captured from female gametocyte or zygote lysates on magnetic streptavidin beads for on-bead tryptic digestion and mass spectrometry analysis.

Evidence is presented that: 'the compositional and spatial rearrangements detected by proximity proteomics are consistent with DOZI/CITH/ALBA mRNPs 1) adopting a condensed state during translational repression in female gametocytes, and then 2) undergoing composition changes that promote its elongation in zygotes following the release from translational repression'.

Additional information

Other mutants