Pyridine nucleotide transhydrogenase (PNT) catalyzes the direct transfer of a 
hydride-ion equivalent between NAD(H) and NADP(H) in bacteria and the 
mitochondria of eukaryotes. PNT was previously postulated to be localized to the 
highly divergent mitochondrion-related organelle, the mitosome, in the 
anaerobic/microaerophilic protozoan parasite Entamoeba histolytica based on the 
potential mitochondrion-targeting signal. However, our previous proteomic study 
of isolated phagosomes suggested that PNT is localized to organelles other than 
mitosomes. An immunofluorescence assay using anti-E. histolytica PNT (EhPNT) 
antibody raised against the NADH-binding domain showed a distribution to the 
membrane of numerous vesicles/vacuoles, including lysosomes and phagosomes. The 
domain(s) required for the trafficking of PNT to vesicles/vacuoles was examined 
by using amoeba transformants expressing a series of carboxyl-terminally 
truncated PNTs fused with green fluorescent protein or a hemagglutinin tag. All 
truncated PNTs failed to reach vesicles/vacuoles and were retained in the 
endoplasmic reticulum. These data indicate that the putative targeting signal is 
not sufficient for the trafficking of PNT to the vesicular/vacuolar compartments 
and that full-length PNT is necessary for correct transport. PNT displayed a 
smear of >120 kDa on SDS-PAGE gels. PNGase F and tunicamycin treatment, chemical 
degradation of carbohydrates, and heat treatment of PNT suggested that the 
apparent aberrant mobility of PNT is likely attributable to its hydrophobic 
nature. PNT that is compartmentalized to the acidic compartments is 
unprecedented in eukaryotes and may possess a unique physiological role in E. 
histolytica.