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Mitochondrial ATP synthase is the terminal complex of oxidative phosphorylation and plays an essential role in parasite energy metabolism. However, the functions of many parasite\u2010specific ATP synthase\u2010associated proteins remain largely unknown. Here, we identified and characterized two novel ATP synthase\u2010associated proteins, TgASAP5 and TgASAP6, in\n                    <jats:italic>T. gondii<\/jats:italic>\n                    and generated conditional knockdown parasite strains to investigate their functions. Subcellular localization and protein interaction analyses demonstrated that TgASAP5 and TgASAP6 are mitochondrial proteins associated with the ATP synthase core subunit F\n                    <jats:sub>1<\/jats:sub>\n                    \u03b2. Functional analyses revealed that depletion of either TgASAP5 or TgASAP6 significantly impaired parasite invasion, intracellular replication, lytic growth, and virulence in mice. Knockdown of TgASAP5 or TgASAP6 disrupted mitochondrial inner membrane organization and was associated with reduced ATP production, elevated reactive oxygen species levels, and reduced mitochondrial membrane potential. Transcriptomic analysis further revealed extensive alterations in mitochondrion\u2010associated gene expression, affecting oxidative phosphorylation and energy metabolism. Collectively, these findings demonstrate that TgASAP5 and TgASAP6 are essential components of the\n                    <jats:italic>T. gondii<\/jats:italic>\n                    ATP synthase complex and are required for maintaining mitochondrial integrity, energy homeostasis, and parasite survival. This study provides new insights into the molecular regulation of mitochondrial ATP synthase in apicomplexan parasites.\n                  <\/jats:p>","DOI":"10.1096\/fj.202603677rr","type":"journal-article","created":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T13:25:20Z","timestamp":1791465920000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["<scp>ATP<\/scp>\n                    Synthase\u2010Associated Proteins\n                    <scp>TgASAP5<\/scp>\n                    and\n                    <scp>TgASAP6<\/scp>\n                    Sustain Mitochondrial Homeostasis in\n                    <scp>\n                      <i>Toxoplasma gondii<\/i>\n                    <\/scp>"],"prefix":"10.1096","volume":"40","author":[{"given":"Xueqin","family":"Zou","sequence":"first","affiliation":[{"name":"Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology Guangxi University  Nanning China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingying","family":"Xue","sequence":"additional","affiliation":[{"name":"Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology Guangxi University  Nanning China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiayi","family":"Huang","sequence":"additional","affiliation":[{"name":"Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology Guangxi University  Nanning China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lijia","family":"Wang","sequence":"additional","affiliation":[{"name":"Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology Guangxi University  Nanning 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