Acetate and hypertonic stress stimulate organelle membrane fission using distinct phosphatidylinositol signals

Abstract

Organelle morphology reflects an equilibrium between membrane fusion and fission that determines size, shape and copy number. By studying the yeast vacuole as a model, the conserved molecular mechanisms responsible for organelle fusion have been revealed. However, a detailed understanding of vacuole fission and how these opposing processes respond to the cell cycle, osmoregulation or metabolism to change morphology remain elusive. Thus, herein we describe a new fluorometric assay to measure vacuole fission in vitro. For proof-of-concept, we use this assay to confirm that acetate, a key intermediary metabolite, triggers vacuole fission in vitro and show that it also blocks homotypic vacuole fusion. The basis of this effect is distinct from hypertonic stress, a known trigger of fission and inhibitor of fusion that inactivates the Rab-GTPase Ypt7: Treatment with the phosphatidylinositol-kinase inhibitor wortmannin or the catalytic domain of the Rab-GAP (GTPase Activating Protein) Gyp1 reveal that fission can be triggered by Ypt7 inactivation alone in absence of hypertonic stress, placing it upstream of PI-3,5-P2 synthesis and osmosis required for membrane scission. Whereas acetate seems to block PI-4-kinase to possibly increase the pool of PI on vacuole membranes needed to synthesize sufficient PI-3,5-P2 for fission. Thus, we speculate that both PI-4-P and PI-3-P arms of PI-P signaling drive changes in membrane fission and fusion responsible altering vacuole morphology in response to cellular metabolism or osmoregulation.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=190 HEIGHT=200 SRC=\"FIGDIR/small/398685_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (28K):\norg.highwire.dtl.DTLVardef@896c6dorg.highwire.dtl.DTLVardef@2d8100org.highwire.dtl.DTLVardef@1702e45org.highwire.dtl.DTLVardef@1eddbba_HPS_FORMAT_FIGEXP M_FIG C_FIG