TY - JOUR
T1 - The Plasmodium falciparum Artemisinin Susceptibility-Associated AP-2 Adaptin μ Subunit is Clathrin Independent and Essential for Schizont Maturation
AU - Henrici, Ryan C.
AU - Edwards, Rachel L.
AU - Zoltner, Martin
AU - van Schalkwyk, Donelly A.
AU - Hart, Melissa N.
AU - Mohring, Franziska
AU - Moon, Robert W.
AU - Nofal, Stephanie D.
AU - Patel, Avnish
AU - Flueck, Christian
AU - Baker, David A.
AU - Odom John, Audrey R.
AU - Field, Mark C.
AU - Sutherland, Colin J.
N1 - Funding Information:
R.C.H. was supported by the UK Foreign and Commonwealth Office through the Marshall Scholarship Program. C.J.S. is supported by Public Health England. M.Z. and M.C.F. are supported by Wellcome Trust 204697/Z/16/Z (to M.C.F.). M.C.F. is a Wellcome Trust Investigator. A.R.O.J. is supported by National Institutes of Health R01 AI103280 and R01 AI123433 and is a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Diseases (PATH). R.W.M. and F.M. are supported by an MRC Career Development Award (MR/M021157/1) jointly funded by the UK Medical Research Council and Department for International Development. M.N.H. is supported by a Bloomsbury Colleges research studentship. We declare there are no competing financial interests.
Publisher Copyright:
© 2020 Henrici et al.
PY - 2020/2
Y1 - 2020/2
N2 - The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.
AB - The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.
KW - Plasmodium falciparum
KW - adaptin trafficking complex
KW - artemisinin susceptibility
KW - adaptor proteins
KW - endocytosis
KW - malaria
KW - Adaptor proteins
KW - Endocytosis
KW - Malaria
KW - Artemisinin susceptibility
KW - Adaptin trafficking complex
UR - http://www.scopus.com/inward/record.url?scp=85079850964&partnerID=8YFLogxK
U2 - 10.1128/mBio.02918-19
DO - 10.1128/mBio.02918-19
M3 - Article
C2 - 32098816
VL - 11
JO - MBio
JF - MBio
SN - 2150-7511
IS - 1
M1 - e02918-19
ER -