Decoding the Minimal Translation System of the Plasmodium falciparum Apicoplast: Essential tRNA-modifying Enzymes and Their Roles in Organelle Maintenance.

Post-transcriptional tRNA modifications are essential for accurate and efficient protein translation across all organisms. The apicoplast organelle genome of Plasmodium falciparum contains a minimal set of 25 complete tRNA isotypes, making it an ideal model for studying minimal translational machinery. Efficient decoding of mRNA codons by this limited tRNA set depends on post-transcriptional modifications. In this study, we sought to define the minimal set of tRNA-modifying enzymes. Using comparative genomics and apicoplast protein localization prediction tools, we identified 16 nucleus-encoded tRNA-modifying enzymes predicted to localize to the apicoplast. Experimental studies confirmed apicoplast localization for 14 enzymes, including two with dual localization. Combining an apicoplast metabolic bypass parasite line with gene disruption tools, we disrupted 12 of the 14 apicoplast-localized enzymes. Six of these enzymes were found to be essential for parasite survival, and six were dispensable. All six essential enzymes are thought to catalyze modifications in the anticodon loop of tRNAs, and their deletions resulted in apicoplast disruption. Of the two genes refractory to deletion, one exhibited dual localization, suggesting essential functions outside the apicoplast. The other, which appears to localize solely to the apicoplast, may play an indispensable role that is not circumvented by our metabolic bypass. Our findings suggest the apicoplast translation system relies on a minimal set of tRNA modifications concentrated in the anticodon loop. This work advances our understanding of minimal translational machinery in reduced organelles, such as the apicoplast, with promising applications in synthetic biology.