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oai:figshare.com:article/34017842 2026-09-28T17:54:54Z category_1 category_4 category_915 category_7 category_8 category_12 category_13 category_132 portal_5 item_type_6 month_year_09_2026

<p>Supplemental methods.</p> Yasmin Schmitz (11974910) Moumita Sengupta (25132859) Carola Schneider (3743791) Tanja Ziesmann (3318936) Franziska Hellmold (25132862) Ute Distler (115774) Rudolph Reimer (190562) Joachim Michael Matz (25132865) Biophysics Biochemistry Physical Sciences not elsewhere classified Medicine Microbiology Cell Biology Genetics Infectious Diseases related protein 18 div >< p broad architectural destabilization balance cargo influx associated protein machinery antimalarial drugs unchanged 3d electron microscopy membrane sector phenocopied endocytic membrane homeostasis endocytic membrane dynamics vacuole constantly fuses specialized digestive vacuole stage parasites hypersensitive vacuolar membrane dynamics efficient hemoglobin processing degrading hemoglobin within asexual parasite proliferation plasmodium falciparum </ atpase assembly factor atg18 links v vacuolar membrane vacuole ’ malaria parasites atpase assembly parasite ’ parasite survival parasite death incoming hemoglobin pf </ vesicle trafficking rendered ring regulatory influence quantitative live proton pump point mutation multifaceted role key regulator interactions indicates interact directly findings establish filled vesicles direct interference decrease sensitivity <div><p>Malaria parasites replicate inside red blood cells, degrading hemoglobin within a specialized digestive vacuole. Efficient hemoglobin processing is essential for parasite survival and influences antimalarial drug susceptibility. The vacuole constantly fuses with incoming hemoglobin-filled vesicles, yet the mechanisms that balance cargo influx with membrane homeostasis remain unclear. Here, using conditional reverse genetics, quantitative live-cell imaging, and 3D electron microscopy, we characterize the autophagy-related protein 18 of <i>Plasmodium falciparum</i> (<i>Pf</i>ATG18) as a key regulator of vacuolar membrane dynamics. Loss of <i>Pf</i>ATG18 caused vacuole fragmentation, accumulation of hemoglobin-filled vesicles, and parasite death. These defects were preceded by broad architectural destabilization of the parasite’s V-ATPase, a proton pump controlling organelle acidification and the vacuole’s fusion–fission equilibrium. Direct interference with its membrane sector phenocopied <i>Pf</i>ATG18 deficiency. We found that <i>Pf</i>ATG18 does not interact directly with the proton pump but instead associates with a putative V-ATPase assembly factor and with complexes regulating phosphoinositide balance and vesicle trafficking. The breadth of these interactions indicates a multifaceted role at the vacuolar membrane and a regulatory influence on V-ATPase mediated through associated protein machinery. Although a point mutation in <i>Pf</i>ATG18 has been linked to artemisinin resistance, its complete knockout did not decrease sensitivity. Instead, it rendered ring-stage parasites hypersensitive to dihydroartemisinin, while leaving their susceptibility to other antimalarial drugs unchanged. Together, these findings establish <i>Pf</i>ATG18 as a central regulator of endocytic membrane homeostasis, essential for V-ATPase function and asexual parasite proliferation in the human blood.</p></div> 2026-09-28T18:08:16Z Text Journal contribution 10.1371/journal.ppat.1014608.s016 https://figshare.com/articles/journal_contribution/_p_Supplemental_methods_p_/34017842 CC BY 4.0