ATP-independent Control of Vac8 Palmitoylation by a SNARE Subcomplex on Yeast Vacuoles

Yeast vacuole fusion requires palmitoylated Vac8. We previously showed that Vac8 acylation occurs early in the fusion reaction, is blocked by antibodies against Sec18 (yeast N-ethylmaleimide-sensitive fusion protein (NSF)), and is mediated by the R-SNARE Ykt6. Here we analyzed the regulation of this reaction on purified vacuoles. We show that Vac8 acylation is restricted to a narrow time window, is independent of ATP hydrolysis by Sec18, and is stimulated by the ion chelator EDTA. Analysis of vacuole protein complexes indicated that Ykt6 is part of a complex distinct from the second R-SNARE, Nyv1. We speculate that during vacuole fusion, Nyv1 is the classical R-SNARE, whereas the Ykt6-containing complex has a novel function in Vac8 palmitoylation

Loss-of-function mutations of SURF-1 are specifically associated with Leigh syndrome with cytochrome c oxidase deficiency

Mutations of SURF-1, a gene located on chromosome 9q34, have recently been identified in patients affected by Leigh syndrome (LS), associated with deficiency of cytochrome c oxidase (COX), the terminal component of the mitochondrial respiratory chain. To investigate to what extent SURF-1 is responsible for human disorders because of COX deficiency, we undertook sequence analysis of the SURF-1 gene in 46 unrelated patients. We analyzed 24 COX-defective patients classified as having typical Leigh syndrome (LSC(COX)), 6 patients classified as Leigh-like (LL(COX)) cases, and 16 patients classified as non-LS(COX) cases. Frameshift, stop, and splice mutations of SURF-1 were detected in 18 of 24 (75%) of the LS(COX) cases. No mutations were found in the LL(COX) and non-LS(COX) group of patients. Rescue of the COX phenotype was observed in transfected cells from patients harboring SURF-1 mutations, but not in transfected cell lines from 2 patients in whom no mutations were detected by sequence analysis. Loss of function of SURF-1 protein is specifically associated with LS(COX), although a proportion of LS(COX) cases must be the result of abnormalities in genes other than SURF-1. SURF-1 is the first nuclear gene to be consistently mutated in a major category of respiratory chain defects. DNA analysis can now be used to accurately diagnose LS(COX), a common subtype of Leigh syndrome

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

Importance of the N-Terminal Domain of the Qb-SNARE Vti1p for Different Membrane Transport Steps in the Yeast Endosomal System

Gossing M, Chidambaram S, Fischer von Mollard G. Importance of the N-Terminal Domain of the Qb-SNARE Vti1p for Different Membrane Transport Steps in the Yeast Endosomal System. Plos One. 2013;8(6): e66304.SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) on transport vesicles and target membranes are crucial for vesicle targeting and fusion. They form SNARE complexes, which contain four a-helical SNARE motifs contributed by three or four different SNAREs. Most SNAREs function only in a single transport step. The yeast SNARE Vti1p participates in four distinct SNARE complexes in transport from the trans Golgi network to late endosomes, in transport to the vacuole, in retrograde transport from endosomes to the trans Golgi network and in retrograde transport within the Golgi. So far, all vti1 mutants investigated had mutations within the SNARE motif. Little is known about the function of the N-terminal domain of Vti1p, which forms a three helix bundle called H-abc domain. Here we generated a temperature-sensitive mutant of this domain to study the effects on different transport steps. The secondary structure of wild type and vti1-3 H-abc domain was analyzed by circular dichroism spectroscopy. The amino acid exchanges identified in the temperature-sensitive vti1-3 mutant caused unfolding of the H-abc domain. Transport pathways were investigated by immunoprecipitation of newly synthesized proteins after pulse-chase labeling and by fluorescence microscopy of a GFP-tagged protein cycling between plasma membrane, early endosomes and Golgi. In vti1-3 cells transport to the late endosome and assembly of the late endosomal SNARE complex was blocked at 37 degrees C. Retrograde transport to the trans Golgi network was affected while fusion with the vacuole was possible but slower. Steady state levels of SNARE complexes mediating these steps were less affected than that of the late endosomal SNARE complex. As different transport steps were affected our data demonstrate the importance of a folded Vti1p H-abc domain for transport

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