A new ATP‐binding cassette protein is involved in intracellular haem trafficking in Leishmania

Abstract

SummaryThe characterization of LABCG5, a new intracellular ATP-binding cassette protein in Leishmania donovani, is described. Unlike other ABCG halftransporters, LABCG5 is not involved in either drug resistance or phospholipid efflux. However, we provide evidence suggesting that this protein is involved in intracellular haem trafficking. Thus, downregulation of LABCG5 function produced upon overexpression of an inactive version of the protein caused a dramatic growth arrest unless a haemin supplement was added or the mutated gene was eliminated. Supplementation with haemoglobin, an upstream metabolite normally sufficient to meet parasite haem requirements, was unable to rescue the growth defect phenotype. Haemoglobin endocytosis was not hampered in dominant-negative parasites and neither was haem uptake, a process that we show here to be dependent on a specific transporter. In contrast, LABCG5 function was required for the correct intracellular trafficking of haemoglobin-bound porphyrins to the mitochondria, not affecting the routing of free haem. Finally, LABCG5 binds haem through hydrophobic and electrostatic interactions. Altogether, these data suggest that LABCG5 is involved in the salvage of the haem released after the breakdown of internalized haemoglobin. As Leishmania is auxotrophic for haem, the pharmacological targeting of this route could represent a novel approach to control fatal visceral leishmaniasis.

A New ABC Half-Transporter in Leishmania major Is Involved in Resistance to Antimony

“…S1 in the supplemental material). In contrast to the phenotype observed after LABCG5 K/M expression (38), parasites transfected with ABCI4 K/M grew at normal rates (data not shown).…”

“…However, although eight subfamilies (ABCA to ABCH) (25) have been identified since the first ABC transporter, which is involved in drug resistance, was described in Leishmania (43), only a limited amount of information regarding the functionality of these proteins has been obtained, mainly as regards drug resistance and lipid/heme trafficking (17,38). In addition, the family of ABC transporters in Leishmania contains an unclassified group of proteins termed as "others" that includes four different transporters with no homology with other eukaryotic ABC proteins, but with orthologues in Trypanosoma brucei and Trypanosoma cruzi (15).…”

“…The dimerization requirement for ABC half-transporters to become functional led us to test a dominant-negative approach to downregulate ABCI4 function, as recently described for Leishmania LABCG5 and LABCG2 (38). To this end, we first mutated the lysine residue inside the Walker A motif in ABCI4 (742 position), which is known to be critical for ATP hydrolysis in ABC transporters (38,39), to a methionine (K742M).…”

“…To this end, we first mutated the lysine residue inside the Walker A motif in ABCI4 (742 position), which is known to be critical for ATP hydrolysis in ABC transporters (38,39), to a methionine (K742M). The expression of other ABCG half-transporters with a similar K/M substitution produces a dominant-negative inhibition in the wild-type transporters (38,40). The resulting construct was transfected into L. major and the expression of ABCI4 K742M (ABCI4 K/M ), as well as other Leishmania lines overexpressing ABCI4 was verified by reverse transcription-PCR (RT-PCR) (see Fig.…”

Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis

“…infantum amastigotes [20] and a specific uptake of the heme analogue magnesium protoporphyrin IX (MgPPIX) in L . donovani [21]. In 2012 a gene in L .…”