Background: Leishmania synthesize pyrimidine nucleotides via biosynthetic and salvage pathways. Results: Genetic blocks in pyrimidine biosynthesis and/or salvage can impact both life cycle stages of Leishmania. Conclusion: Pyrimidine biosynthesis and salvage both contribute to Leishmania homeostasis in animal infections. Significance: Functional characterization of pyrimidine pathways is crucial for understanding pyrimidine metabolism and validation of potential drug targets and vaccine strains.

Lysosomes are dynamic organelles with critical roles in cellular physiology. The lysosomal signaling lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is a key regulator that has been implicated to control lysosome ion homeostasis, but the scope of ion transporters targeted by PI(3,5)P2 and the purpose of this regulation is not well understood. Through an unbiased screen in Saccharomyces cerevisiae, we identified loss-of-function mutations in the vacuolar H+-ATPase (V-ATPase) and in Vnx1, a vacuolar monovalent cation/proton antiporter, as suppressor mutations that relieve the growth defects and osmotic swelling of vacuoles (lysosomes) in yeast lacking PI(3,5)P2. We observed that depletion of PI(3,5)P2 synthesis in yeast causes a robust accumulation of multiple cations, most notably an ∼85 mM increase in the cellular concentration of potassium, a critical ion used by cells to regulate osmolarity. The accumulation of potassium and other cations in PI(3,5)P2-deficient yeast is relieved by mutations that inactivate Vnx1 or inactivate the V-ATPase and by mutations that increase the activity of a vacuolar cation export channel, Yvc1. Collectively, our data demonstrate that PI(3,5)P2 signaling orchestrates vacuole/lysosome cation transport to aid cellular osmoregulation.

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We present solution-state NMR structures for two putative venom peptides from Sicarius dolichocephalus. These peptides were identified from cDNA libraries created from venom gland mRNA and then recombinantly expressed. They are the first structures from any species of Sicarius spiders, and the first peptide structures for any haplogyne spiders. These peptides are homologous to one another, and while they have at most only 20% sequence identity with known venom peptides their structures follow the inhibitor cystine knot motif that has been found in a broad range of venom peptides.

Endosomes undergo a maturation process highlighted by a reduction in lumenal pH, a conversion of surface markers that prime endosome-lysosome fusion, and the sequestration of ubiquitinated transmembrane protein cargoes within intralumenal vesicles (ILVs). We investigated ILV cargo sorting in mutant strains of the budding yeast Saccharomyces cerevisiae that are deficient for either the lysosomal/vacuolar signaling lipid PI(3,5)P2 or the Doa4 ubiquitin hydrolase that deubiquitinates ILV cargoes. Disruption of PI(3,5)P2 synthesis or Doa4 function causes a defect in the sorting of a subset of ILV cargoes. We show that these cargo-sorting defects are suppressed by mutations that disrupt Vph1, which is a subunit of Vacuolar H+-ATPase (V-ATPase) complexes that acidify late endosomes and vacuoles. We further show that Vph1 dysfunction increases endosome abundance and disrupts vacuolar localization of Ypt7 and Vps41, two critical mediators of endosome-vacuole fusion. Because V-ATPase inhibition attenuates endosome-vacuole fusion and rescues the ILV cargo-sorting defects in yeast lacking PI(3,5)P2 and Doa4 activity, our results suggest that the V-ATPase performs a role in the coordination of ILV cargo sorting with the membrane fusion machinery.