LdARF1 in trafficking and structural maintenance of the trans-Golgi cisternal network in the protozoan pathogen Leishmania donovani.

Abstract

Adenosine diphosphate ribosylation factors (ARFs) are small guanosine-5'-triphosphatases that are essential in vesicular trafficking and in the maintenance of the Golgi network. In this report, we identified a homolog of the mammalian ARF1 in the human pathogenic protozoan parasite, Leishmania donovani (Ld). Ld ARF1 is a 549 bp gene encoding a 183-amino acid deduced protein of approximately 20 kDa. We demonstrated by Southern blot analysis that there are at least two copies of ARF1 in the Ld genome. Moreover, Northern blot analysis revealed that Ld ARF1 is expressed on a 1.35 kb transcript in both the insect vector (promastigotes) and mammalian host (amastigotes) forms of this parasite. Fluorescent microscopy studies using Ld promastigotes episomally transfected with an ARF1::GFP (green fluorescent protein) chimeric construct showed that such chimeras appeared to localize to the Golgi region of these organisms. This observation was verified by immunoelectron microscopy using an anti-GFP antibody. Such studies also revealed that Ld ARF1::GFP chimeras localized to trans-Golgi vesicles, the flagellar pocket/reservoir and other vesicles located between the trans-Golgi network and flagellar pocket in these apically polarized cells. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching experiments revealed both the dynamic binding and releasing activity of Ld ARF1 from the Golgi network in these parasites. Further, episomal expression of a constitutively active ("on") ARF1 (Q71L mutation) resulted in the aberrant swelling and distended-structure of the trans-Golgi cisternae in these cells. These results show that Ld ARF1 is transiently associated with the Golgi network and plays a role in the structural maintenance of this organelle in these important human pathogens.