Functional screening reveals Toxoplasma prenylated proteins required for endocytic trafficking and rhoptry protein sorting

Wang, Qiang-Qiang; Sun, Ming; Tang, Tao; Lai, De-Hua; Liu, Jing; Maity, Sanjay; He, Kai; Wu, Xi-Ting; Yang, Jiong; Li, Yue-Bao; Tang, Xiao-Yan; Ding, Hui-Yong; Hide, Geoff; Distefano, Mark; Lun, Zhao-Rong; Zhu, Xing-Quan; Long, Shaojun

Abstract

In the apicomplexans, endocytosed cargos (e.g., hemoglobin) are trafficked to a specialized organelle for digestion. This follows a unique endocytotic process at the micropore/cytostome in these parasites. However, the mechanism underlying endocytic trafficking remains elusive, due to the repurposing of classical endocytic proteins for the biogenesis of apical organelles. To resolve this issue, we have exploited the genetic tractability of the model apicomplexan Toxoplasma gondii, which ingests host cytosolic materials (e.g., green fluorescent protein[GFP]). We determined an association between protein prenylation and endocytic trafficking, and using an alkyne-labeled click chemistry approach, the prenylated proteome was characterized. Genome editing, using clustered regularly interspaced short palindromic repaet/CRISPR-associated nuclease 9 (CRISPR/Cas9), was efficiently utilized to generate genetically modified lines for the functional screening of 23 prenylated candidates. This identified four of these proteins that regulate the trafficking of endocytosed GFP vesicles. Among these proteins, Rab1B and YKT6.1 are highly conserved but are non-classical endocytic proteins in eukaryotes. Confocal imaging analysis showed that Rab1B and Ras are substantially localized to both the trans-Golgi network and the endosome-like compartments in the parasite. Conditional knockdown of Rab1B caused a rapid defect in secretory trafficking to the rhoptry bulb, suggesting a trafficking intersection role for the key regulator Rab1B. Further experiments confirmed a critical role for protein prenylation in regulating the stability/activity of these proteins (i.e., Rab1B and YKT6.1) in the parasite. Our findings define the molecular basis of endocytic trafficking and reveal a potential intersection function of Rab1B on membrane trafficking in T. gondii. This might extend to other related protists, including the malarial parasites.

Citation

Wang, Q., Sun, M., Tang, T., Lai, D., Liu, J., Maity, S., …Long, S. (2023). Functional screening reveals Toxoplasma prenylated proteins required for endocytic trafficking and rhoptry protein sorting. mBio, https://doi.org/10.1128/mbio.01309-23

• Journal Article Type: Article
• Acceptance Date: Jun 12, 2023
• Online Publication Date: Aug 7, 2023
• Publication Date: Aug 7, 2023
• Deposit Date: Aug 9, 2023
• Publicly Available Date: Aug 14, 2023
• Journal: mBio
• Print ISSN: 2150-7511
• Publisher: American Society for Microbiology
• Peer Reviewed: Peer Reviewed
• DOI: https://doi.org/10.1128/mbio.01309-23
• Keywords: Virology; Microbiology