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Upon entering the mosquito midgut, the gametocytes egress from the enveloping erythrocyte while passing through gametogenesis. Egress follows an inside\u2010out mode during which the membrane of the parasitophorous vacuole (PV) ruptures prior to the erythrocyte membrane. Membrane rupture requires exocytosis of specialized egress vesicles of the parasites; that is, osmiophilic bodies (OBs) involved in rupturing the PV membrane, and vesicles that harbor the perforin\u2010like protein PPLP2 (here termed P\u2010EVs) required for erythrocyte lysis. While some OB proteins have been identified, like G377 and MDV1\/Peg3, the majority of egress vesicle\u2010resident proteins is yet unknown. Here, we used high\u2010resolution imaging and BioID methods to study the two egress vesicle types in <jats:italic>Plasmodium falciparum<\/jats:italic> gametocytes. We show that OB exocytosis precedes discharge of the P\u2010EVs and that exocytosis of the P\u2010EVs, but not of the OBs, is calcium sensitive. Both vesicle types exhibit distinct proteomes with the majority of proteins located in the OBs. In addition to known egress\u2010related proteins, we identified novel components of OBs and P\u2010EVs, including vesicle\u2010trafficking proteins. Our data provide insight into the immense molecular machinery required for the inside\u2010out egress of <jats:italic>P. falciparum<\/jats:italic> gametocytes.<\/jats:p>","DOI":"10.1111\/mmi.15125","type":"journal-article","created":{"date-parts":[[2023,7,26]],"date-time":"2023-07-26T09:21:12Z","timestamp":1690363272000},"page":"431-452","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Comparative proteomics of vesicles essential for the egress of <i>Plasmodium falciparum<\/i> gametocytes from red blood cells"],"prefix":"10.1111","volume":"121","author":[{"given":"Juliane","family":"Sassmannshausen","sequence":"first","affiliation":[{"name":"Division of Cellular and Applied Infection Biology Institute of Zoology, RWTH Aachen University  Aachen Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra","family":"Bennink","sequence":"additional","affiliation":[{"name":"Division of Cellular and Applied Infection Biology Institute of Zoology, RWTH Aachen University  Aachen Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ute","family":"Distler","sequence":"additional","affiliation":[{"name":"Core Facility for Mass Spectrometry Institute of Immunology, University Medical Centre of the Johannes\u2010Gutenberg University  Mainz Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juliane","family":"K\u00fcchenhoff","sequence":"additional","affiliation":[{"name":"Division of Cellular and Applied Infection Biology Institute of Zoology, RWTH Aachen University  Aachen Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Allen M.","family":"Minns","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Huck Center for Malaria Research The Pennsylvania State University  University Park Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Scott E.","family":"Lindner","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Huck Center for Malaria Research The Pennsylvania State University  University Park Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0461-4352","authenticated-orcid":false,"given":"Paul\u2010Christian","family":"Burda","sequence":"additional","affiliation":[{"name":"Centre for Structural Systems Biology  Hamburg Germany"},{"name":"Bernhard Nocht Institute for Tropical Medicine  Hamburg Germany"},{"name":"Biology Department University of Hamburg  Hamburg Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefan","family":"Tenzer","sequence":"additional","affiliation":[{"name":"Core Facility for Mass Spectrometry Institute of Immunology, University Medical Centre of the Johannes\u2010Gutenberg University  Mainz 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