title: Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1.
abstract: Size and copy number of organelles are influenced by an equilibrium of membrane fusion and fission. We studied this equilibrium on vacuoles-the lysosomes of yeast. Vacuole fusion can readily be reconstituted and quantified in vitro, but it had not been possible to study fission of the organelle in a similar way. Here we present a cell-free system that reconstitutes fragmentation of purified yeast vacuoles (lysosomes) into smaller vesicles. Fragmentation in vitro reproduces physiological aspects. It requires the dynamin-like GTPase Vps1p, V-ATPase pump activity, cytosolic proteins, and ATP and GTP hydrolysis. We used the in vitro system to show that the vacuole-associated TOR complex 1 (TORC1) stimulates vacuole fragmentation but not the opposing reaction of vacuole fusion. Under nutrient restriction, TORC1 is inactivated, and the continuing fusion activity then dominates the fusion/fission equilibrium, decreasing the copy number and increasing the volume of the vacuolar compartment. This result can explain why nutrient restriction not only induces autophagy and a massive buildup of vacuolar/lysosomal hydrolases, but also leads to a concomitant increase in volume of the vacuolar compartment by coalescence of the organelles into a single large compartment.
authors.affiliation: Département de Biochimie, Université de Lausanne, 1066 Epalinges, Switzerland.
authors.forename: Lydie
authors.initials: L
authors.lastname: Michaillat
authors.forename: Tonie Luise
authors.initials: TL
authors.lastname: Baars
authors.forename: Andreas
authors.initials: A
authors.lastname: Mayer
mesh_terms: D002474:Cell-Free System / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D008561:Membrane Fusion; D054648:Protein Phosphatase 2 / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014617:Vacuoles / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: D029701:Saccharomyces cerevisiae Proteins; C561842:TORC1 protein complex, S cerevisiae; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; D006160:Guanosine Triphosphate; D054648:Protein Phosphatase 2; C104343:SIT4 protein, S cerevisiae; D019204:GTP-Binding Proteins; C064553:VPS1 protein, S cerevisiae; D020123:Sirolimus