Phenotypical characterization of novel apicoplast membrane proteins in Toxoplasma gondii

Abstract As a critical organelle in Toxoplasma gondii , an important zoonotic pathogen, the apicoplast plays a vital role in survival by hosting essential metabolic pathways. Its unique four-membrane structure provides both stability and a relatively independent metabolic compartment. To identify previously uncharacterized apicoplast membrane proteins, we undertook a proteomics analysis of the apicoplast using a TurboID proximity biotinylation approach with our previously discovered apicoplast membrane proteins—AMT1, AMT2, HAP1, and HAP9. The identified apicoplast membrane proteins in this study, along with 10 previously discovered apicoplast membrane proteins, were subjected to phenotypic and functional analyses. This demonstrated that the major facilitator superfamily transport HAP1 and the ATP-binding cassette transporter HAP9 are both apicoplast proteins essential for in vitro parasite growth in host cell monolayers and in vivo mouse infection. Depletion of the transporters caused similar defects in parasite replication, endogenous biotinylation of ACC1 and stability of the apicoplast. However, transmission electron microscopy showed that depletion of HAP1 and HAP9 caused different structural defects in the apicoplast: a lucent intra-membrane space in the HAP1-depleted apicoplast, and a lucent matrix and loosened membranes in the HAP9 depleted apicoplast. This study thus provides critical insights into the physiological and structural roles of these newly identified transporters in the apicoplast of T. gondii and, potentially, in closely related protists containing the apicoplast.