Phosphatidylinositol 3,5‐Bisphosphate acts as a Novel Regulator of Calcium Transport During Saccharomyces Cerevisiae vacuolar fusion

Abstract

Phosphoinositides (PIs) are a group of lipids that are differentially phosphorylated to provide specific protein‐lipid interactions that are necessary in maintaining eukaryotic homeostasis. In membrane traffic, different PIs can interact with nucleotide exchange factors, tethering molecules and SNARE proteins ultimately leading to membrane fusion and transfer of cargo material. Our work has shown that phosphatidylinositol 3‐phosphate (PI3P), PI4P and PI(4,5)P2 are essential for the fusion of yeast vacuoles/lysosomes. However, little is known about the role of PI(3,5)P2 in the fusion pathway. Others have shown that PI(3,5)P2 is necessary for fission to occur during osmotic shock that is coupled to the efflux of vacuolar Ca2+ into the cytoplasm. This is thought to occur through the interaction of PI(3,5)P2 with the yeast TRP Ca2+ channel (Yvc1). Ca2+ efflux has also been linked to vacuole fusion and occurs upon the formation of SNARE complexes between partner vesicles. Our data show that increasing levels of PI(3,5)P2 (through the addition of exogenous short chain (diC8) lipid) led to inhibition of vacuolar fusion. Importantly, sub‐inhibitory concentrations of diC8‐PI(3,5)P2 completely blocked Ca2+ efflux without affecting fusion. This suggests that PI(3,5)P2 plays opposing roles in fission, fusion, and Ca2+ transport. It further suggests that the Ca2+ efflux channel used during fusion is different than Yvc1. Finally, these data indicate that Ca2+ efflux is not required for vacuolar fusion and may be a parallel response. To further elucidate the role of PI(3,5)P2 in fusion we examined the effect of inhibiting the PI3P 5‐kinase, Fab1 using the PIKfyve (human homolog of Fab1) inhibitor Apilimod. In accord with our other data, treatment of vacuoles with Apilimod led to enhanced Ca2+ efflux, illustrating that PI(3,5)P2 negatively regulates fusion‐linked Ca2+ efflux, however the mechanism through which this occurs remains unknown. In addition to effects on Ca2+ transport, PI(3,5)P2 has been shown to enhance vacuolar H+ pumping through its association with the V‐ATPase. This is further supported by our data showing that Apilimod inhibited H+ pumping. Taken together we now hypothesize that PI(3,5)P2 may affect the activity of the H+/Ca2+ exchanger, Vcx1, due to its effect on both H+ pumping and Ca2+ efflux.Support or Funding InformationNIGMS GM101132