Identification and sub-cellular localization of a NAD transporter in Leishmania braziliensis (LbNDT1)

Abstract

Nicotinamide adenine dinucleotide (NAD) is one of the central molecules involved in energy homeostasis, cellular signaling and antioxidative defense systems. Consequently, its biosynthetic pathways and transport systems are of vital importance. The nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT), a key enzyme in the biosynthesis of NAD, is distributed in all domains of life and exhibits various isoforms in free-living organisms in contrast with intracellular parasites, which displays a single enzyme. In Leishmania braziliensis a unique cytosolic NMNAT has been reported to date and the mechanisms through which adequate levels of NAD are maintained among the different sub-cellular compartments of this parasite are unknown. Experimental evidences have related the transport of NAD to the Nucleotide Transporters (NTTs) family, whose members are located in the cytoplasmic membrane of parasitic life organisms. Additionally, the Mitochondrial Carrier Family (MCF), a group of proteins located in the membrane of internal organelles such as the mitochondria of free life organisms, has been implicated in NAD transport. Applying bioinformatics tools, the main characteristics of the MCF were found in a transporter candidate that we have designated as Nicotinamide Adenine Dinucleotide Transporter 1 of L. braziliensis (LbNDT1). The expression of LbNDT1 was tested both in axenic amastigotes and promastigotes of L. braziliensis, through immunodetection using polyclonal avian antibodies produced in this study. N-glycosylation of LbNDT1 was observed in both stages. Additionally, a possible partial mitochondrial distribution for LbNDT1 in amastigotes and a possible glycosomal location in promastigotes are proposed. Finally, the capability of LbNDT1 to transport NAD was confirmed by complementation assays in Saccharomyces cerevisiae. Our results demonstrate the existence of LbNDT1 in L. braziliensis becoming the first NAD transporter identified in protozoan parasites to date.