Functional role of evolutionarily highly conserved residues, N-glycosylation level and domains of the Leishmania miltefosine transporter-Cdc50 subunit

Abstract

Cdc50 (cell-cycle control protein 50) is a family of conserved eukaryotic proteins that interact with P4-ATPases (phospholipid translocases). Cdc50 association is essential for the endoplasmic reticulum export of P4-ATPases and proper translocase activity. In the present study, we analysed the role of Leishmania infantum LiRos3, the Cdc50 subunit of the P4-ATPase MLF (miltefosine) transporter [LiMT (L. infantum MLF transporter)], on trafficking and complex functionality using site-directed mutagenesis and domain substitution. We identified 22 invariant residues in the Cdc50 proteins from L. infantum, human and yeast. Seven of these residues are found in the extracellular domain of LiRos3, the conservation of which is critical for ensuring that LiMT arrives at the plasma membrane. The substitution of other invariant residues affects complex trafficking to a lesser extent. Furthermore, invariant residues located in the N-terminal cytosolic domain play a role in the transport activity. Partial N-glycosylation of LiRos3 reduces MLF transport and total N-deglycosylation completely inhibits LiMT trafficking to the plasma membrane. One of the N-glycosylation residues is invariant along the Cdc50 family. The transmembrane and exoplasmic domains are not interchangeable with the other two L. infantum Cdc50 proteins to maintain LiMT interaction. Taken together, these findings indicate that both invariant and N-glycosylated residues of LiRos3 are implicated in LiMT trafficking and transport activity.

A P4-ATPase subunit of the Cdc50 family plays a role in iron acquisition and virulence inCryptococcus neoformans

“…neoformans (Widmer et al, ). The drug is an alkylphospholipid that is internalized by P4‐ATPase activity in Leishmania (Garcia‐Sanchez, Sanchez‐Canete, Gamarro, & Castanys, ; Hanson et al, ; Ravu et al, ; Widmer et al, ). Surprisingly, deletion of CDC50 in C .…”

“…neoformans also increased sensitivity to drugs that reflect changes in the asymmetrical distribution or movement of phospholipids in membranes such as papuaminde A (PS; Huang et al, ), cinnamycin (phosphatidlyethanolamine), and miltefosine (phosphatidylcholine). Miltefosine is particularly interesting because this alkylphosphocholine drug is used to treat protozoal and fungal diseases (Garcia‐Sanchez et al, ; Hanson et al, ; Ravu et al, ; Widmer et al, ). It is curious that cdc50 or lem3 mutants in S .…”

On the molecular mechanism of flippase- and scramblase-mediated phospholipid transport

“…Exchanging the N-terminal portion of CDC50A preceding the first transmembrane span with that of CDC50B did not prevent ATP8A2 to interact with CDC50A but substantially decreased its NBD-PS flipping activity after purification of the complex, supporting a role for CDC50 proteins in P4-ATPase-mediated phospholipid transport [111]. A pivotal role of the cytosolic N-terminus of the Leishmania Cdc50 subunit LiRos3 in miltefosine uptake (a substrate of LiMT, a Leishmania infantum P4-ATPase) was also inferred from site-directed mutagenesis of strongly conserved residues [119]. In another study, Takahashi et al investigated the contribution of Cdc50p to the in vivo function of Drs2p [118].…”

Drug resistance in protozoan parasites

“…For example, in Leishmania the miltefosine transporter is a phospholipid translocase [35] and methotrexate is taken up by a folate transporter [13]; the T. brucei carrier for eflornithine is an amino acid transporter [17], while pentamidine and melarsoprol are taken up by an aminopurine transporter and an aquaglyceroporin [18,21]. Thus, it is often hard to predict what might be a ( potential) drug transporter in a protozoan: they are hidden in plain sight, masked by their innocuous roles.…”