Phospholipid translocation and miltefosine potency require both L. donovani miltefosine transporter and the new protein LdRos3 in Leishmania parasites.
The antitumor drug miltefosine has been recently approved as the first oral drug active against visceral leishmaniasis. We have previously identified the L. donovani miltefosine transporter (LdMT) as a P-type ATPase involved in phospholipid translocation at the plasma membrane of Leishmania parasites. Here we show that this protein is essential but not sufficient for the phospholipid translocation activity and, thus, for the potency of the drug. Based on recent findings in yeast, we have identified the putative beta subunit of LdMT, named LdRos3, as another protein factor required for the translocation activity. LdRos3 belongs to the CDC50/Lem3 family, proposed as likely beta subunits for P4-ATPases. The phenotype of LdRos3-defective parasites was identical to that of the LdMT-/-, including a defect in the uptake of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)-phosphatidylserine, generally considered as not affected in Lem3p-deficient yeast. Both LdMT and LdRos3 normally localized to the plasma membrane but were retained inside the endoplasmic reticulum in the absence of the other protein or when inactivating point mutations were introduced in LdMT. Modulating the expression levels of either protein independently, we show that any one of them could behave as the protein limiting the level of flippase activity. Thus, LdMT and LdRos3 seem to form part of the same translocation machinery that determines flippase activity and miltefosine sensitivity in Leishmania, further supporting the consideration of CDC50/Lem3 proteins as beta subunits required for the normal functioning of P4-ATPases.
Pérez-Victoria
F Javier
FJ
Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Avda. del Conocimiento s/n 18100 Armilla, Granada, Spain.
Sánchez-Cañete
María P
MP
Castanys
Santiago
S
Gamarro
Francisco
F
D000251:Adenosine Triphosphatases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D001692:Biological Transport; D018390:Gene Targeting; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007893:Leishmania donovani / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D021122:Protein Subunits / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D026901:Membrane Transport Proteins; D010743:Phospholipids; D021122:Protein Subunits; D015800:Protozoan Proteins; D010767:Phosphorylcholine; D049452:Green Fluorescent Proteins; C039128:miltefosine; D000251:Adenosine Triphosphatases; C480230:miltefosine transporter, Leishmania donovani