7 research outputs found
The HOPS/class C Vps complex tethers membranes by binding to one Rab GTPase in each apposed membrane
Identification of <i>Drosophila</i> Gene Products Required for Phagocytosis of <i>Leishmania donovani</i>
<div><p>The identity and function of host factors required for efficient phagocytosis and intracellular maintenance of the protozoan parasite <i>Leishmania donovani</i> are poorly understood. Utilising the phagocytic capability of <i>Drosophila</i> S2 cells, together with available tools for modulating gene expression by RNAi, we have developed an experimental system in which to identify host proteins of this type on a genome-wide scale. We have shown that <i>L. donovani</i> amastigotes can be phagocytosed by S2 cells, in which they replicate and are maintained in a compartment with features characteristic of mammalian phagolysosomes. Screening with dsRNAs from 1920 conserved metazoan genes has identified transcripts that, when reduced in expression, cause either increased or decreased phagocytosis. Focussing on genes in the latter class, RNAi-mediated knockdown of the small GTPase Rab5, the prenylated SNARE protein YKT6, one sub-unit of serine palmitoyltransferase (<i>spt2/lace),</i> the Rac1-associated protein Sra1 and the actin cytoskeleton regulatory protein, SCAR, all lead to a significant reduction in parasite phagocytosis. A role for the <i>lace</i> mammalian homologue in amastigote uptake by mammalian macrophages has been verified using the serine palmitoyltransferase inhibitor, myriocin. These observations suggest that this experimental approach has the potential to identify a large number of host effectors required for efficient parasite uptake and maintenance.</p></div
PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex
ER-Golgi membrane transport and autophagy are intersecting trafficking pathways that are tightly regulated and crucial for homeostasis, development and diseases. Here, we identify UVRAG, a Beclin1-binding autophagic factor, as a PI(3)P-binding protein that depends on PI(3)P for its ER localization. We further show that UVRAG interacts with RINT-1, and acts as an integral component of the RINT-1-containing ER tethering complex, which couples phosphoinositide metabolism to COPI-vesicle tethering. Displacement or knockdown of UVRAG profoundly disrupted COPI cargo transfer to the ER and Golgi integrity. Intriguingly, autophagy caused the dissociation of UVRAG from the ER tether, which in turn worked in concert with the Bif-1-Beclin-PI(3)KC3 complex to mobilize Atg9 translocation for autophagosome formation. These findings identify a regulatory mechanism that coordinates Golgi-ER retrograde and autophagy-related vesicular trafficking events through physical and functional interactions between UVRAG, phosphoinositide, and their regulatory factors, thereby ensuring spatiotemporal fidelity of membrane trafficking and maintenance of organelle homeostasis