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J. Biol. Chem., Vol. 281, Issue 33, 23766-23775, August 18, 2006

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Phospholipid Translocation and Miltefosine Potency Require Both L. donovani Miltefosine Transporter and the New Protein LdRos3 in Leishmania Parasites^*

From the Instituto de Parasitología y Biomedicina "López-Neyra," Consejo Superior de Investigaciones Científicas, Avda. del Conocimiento s/n 18100 Armilla, Granada, Spain

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 subunit of LdMT, named LdRos3, as another protein factor required for the translocation activity. LdRos3 belongs to the CDC50/Lem3 family, proposed as likely 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 subunits required for the normal functioning of P₄-ATPases.

Received for publication, May 31, 2006 , and in revised form, June 13, 2006.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-Bank^TM/EBI Data Bank with accession number(s) DQ205096 [GenBank] .

^* This work was supported by Fondo de Investigación Sanitaria (FIS) Network Red de Investigación de Centros de Enfermedades Tropicales (RICET) C03-04 (to F. G.), European Community Grant QLRT-2000-01404 (to F. G.), European Community Marie Research Training Network Grant MRTN-CT-2004-005330 (to F. G.), Acciones Integradas con Alemania Grant HA2003-166 (to S. C.), and Spanish Grant SAF2005-01639 (to S. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ These authors contributed equally to this work.

² Recipient of a fellowship from the Ministerio de Educación y Cultura.

³ Senior investigators in this study.

⁴ To whom correspondence should be addressed. Tel.: 34-958-181667; Fax: 34-958-181632; E-mail: gamarro{at}ipb.csic.es.

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