The minimal vesicular trafficking machinery of Giardia has two NSF paralogues.

Vesicle fusion events are crucial for the survival of Giardia lamblia as they drive nutrient uptake and morphological stage transitions. Unlike most eukaryotes, Giardia has a minimal vesicular trafficking machinery. We report a rare exception to this minimalism wherein two paralogues of N-ethylmaleimide-sensitive factor (NSF) are present in this parasite. Localization studies indicate that these highly homologous paralogues-GlNSF112681 and GlNSF114776-likely function independently under various stress conditions, as GlNSF112681 remains at peripheral vesicles, while the major pool of GlNSF114776 redistributes to anterior flagella-associated structures. These paralogues also exhibit selective affinity for the α-soluble NSF attachment proteins (Glα-SNAPs). This selectivity stems from sequence divergences near their N termini. The two GlNSFs colocalize and coimmunoprecipitate, indicating the presence of a heterohexameric 20S complex in trophozoites. This study is the first to report the presence of a heterohexameric 20S complex and reveals adaptive specialization of vesicle trafficking machinery within a reduced eukaryotic system. Impact statement Here we report that a unicellular parasitic protist, Giardia lamblia, has two NSF paralogues, which is a rarity in eukaryotes. Although they share a high degree of homology, they are likely to discharge independent functions, especially under stress conditions.