The Leishmania ARL-1 and Golgi Traffic

Abstract

We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.

Trafficking activity of myosin XXI is required in assembly ofLeishmaniaflagellum

“…6), suggesting a defect in vesicular transport (Garcia-Salcedo et al, 2004;Allen et al, 2003) in these cells. To confirm this observation, we analyzed the transport of endocytic vesicles in Leishmania promastigotes by using the fluorophore N-(3-triethylammoniumpropyl)-4-{6-[4-(diethylamino)phenyl]-hexatrienyl}pyridinium dibromide (FM4-64), which has been widely used as a marker for assessing the flagellar-pocket activity in trypanosomatids (Sahin et al, 2008;Mullin et al, 2001). In wild-type cells, flagellar pockets were typically marked at 4°C, and the fluorophore efficiently endocytosed and trafficked down to form a characteristic multivesicular tubule (Ghedin et al, 2001) at 25°C within 1 hour (Fig.…”

Roles of N-Terminal Fatty Acid Acylations in Membrane Compartment Partitioning:Arabidopsis h-Type Thioredoxins as a Case Study

“…It is interesting to notice that, for few of them, the N-terminal MYR alone without any evident second signal has been shown to target these proteins to intracellular membrane compartments with the ER/Golgi complex being the most frequent localization observed (Resh, 2006). For instance, this is the case for proteins such as eNos (Liu et al, 1997), GRASP (Kondylis et al, 2005), and the Arl family (Price et al, 2005;Sahin et al, 2008). Relocalization at the ER/Golgi compartment from the PM was also observed when the second signal of several known MYRed proteins was abolished, as in the Fyn and Yes protein kinases, in which the Cys residues necessary for the PAL were substituted by a Ser or the polybasic domain of Src protein kinase was made shorter (McCabe and Berthiaume, 1999).…”

ADF/cofilin-driven actin dynamics in early events ofLeishmaniacell division

“…The flagellar pockets appeared enlarged, and a large number of membrane-bound vesicles were seen to accumulate close to the lumen of the flagellar pocket as well as in the cell body, suggesting disturbances in membrane trafficking in the COF -/-cells. To determine whether the flagellar pockets were still functional, we assessed the endocytic activity using the fluorophore N-(3-triethylammoniumpropyl)-4-{6-[4-(diethylamino)phenyl]-hexatrienyl} pyridinium dibromide (FM4-64), which has been widely used as a marker for assessing the flagellar pocket activity in trypanosomatids (Sahin et al, 2008;Mullin et al, 2001). In Leishmania, FM4-64 is readily internalized and targeted through endosomes to the final digestive compartment towards the posterior end (Waller and McConville, 2002) S4) and its redistribution occurred only at the later stages of cell division.…”

“…For monitoring vesicular trafficking activity, internalization of FM4-64 was performed essentially as described (Sahin et al, 2008). Briefly, 10 7 /ml exponentially growing cells were incubated in DMEM containing 10% FCS and 2 g/ml FM4-64 (Molecular Probes) dye was added and the mixture incubated at 4°C for 15 minutes and thereafter the temperature was raised to 25°C.…”