Identification and Characterization of a Polyamine Permease from the Protozoan Parasite Leishmania major

Abstract

The proteins that mediate polyamine translocation into eukaryotic cells have not been identified at the molecular level. To define the polyamine transport pathways in eukaryotic cells we have cloned a gene, LmPOT1, that encodes a polyamine transporter from the protozoan pathogen, Leishmania major. Sequence analysis of LmPOT1 predicted an unusual 803-residue polytopic protein with 9 -12 transmembrane domains. Expression of LmPOT1 cRNA in Xenopus laevis oocytes revealed LmPOT1 to be a high affinity transporter for both putrescine and spermidine, whereas expression of LmPOT1 in Trypanosoma brucei stimulated putrescine uptake that was sensitive to inhibition by pentamidine and proton ionophores. Immunoblot analysis established that LmPOT1 was expressed predominantly in the insect vector form of L. major, and immunofluorescence demonstrated that LmPOT1 was localized predominantly to the parasite plasma membrane. To our knowledge this is the first molecular identification and characterization of a cell surface polyamine transporter in eukaryotic cells.

The putrescine analogue 1,4-diamino-2-butanone affects polyamine synthesis, transport, ultrastructure and intracellular survival in Leishmania amazonensis

“…In addition, pentamidine resistance in Leishmania involves drug extrusion from the mitochondria (Basselin et al, 2002). In this regard, the PA transporter LmPOTI of Leishmania major is inhibited by pentamidine (Hasne & Ullman, 2005). In addition putrescine may stabilize T. cruzi mitochondrial membranes (Giffin et al, 1986).…”

“…Leishmania parasites not only synthesize putrescine and spermidine, but also accumulate extracellular PAs via specific transport systems. Energy-dependent putrescine and spermidine transporters have been described in L. donovani, L. mexicana (Basselin et al, 2000) and L. major (Hasne & Ullman, 2005). PA transport in protozoa may furnish potential targets for parasitic disease chemotherapy (Reguera et al, 2005).…”

“…PA transport in protozoa may furnish potential targets for parasitic disease chemotherapy (Reguera et al, 2005). A PA permease was identified and characterized in L. major (Hasne & Ullman, 2005). These authors showed that the protein, termed LmPOT1, transports putrescine and spermidine, and transfected in T. brucei can stimulate putrescine uptake.…”

AGP2 Encodes the Major Permease for High Affinity Polyamine Import in Saccharomyces cerevisiae

“…Along with LmPot1 from L. major (6) and Gap1p from S. cerevisiae (14), Agp2p is the first eukaryotic permease reported exhibiting high affinity polyamine import across the plasma membrane. As expected from the fact that they both belong to the YAT permease family, Agp2p and Gap1p share extensive homology (28% identity, 59% similarity) (Fig.…”

“…On the other hand, much less is known about the molecular identity of polyamine carrier proteins in eukaryotic cells. During the preparation of this manuscript, a putrescine-preferential plasma membrane permease catalyzing the influx of putrescine and spermidine has been identified in the parasite Leishmania major (6). In Saccharomyces cerevisiae, four H ϩ gradient-dependent Tpo permeases (Tpo1p, Tpo2p, Tpo3p, and Tpo4p) belonging to the H ϩ /drug antiporter subfamily with 12 transmembrane spans (7) catalyze the efflux of polyamines as well as a large variety of organic substrates (e.g.…”