Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1
- 1 March 2012
- journal article
- Published by American Society for Cell Biology (ASCB) in Molecular Biology of the Cell
- Vol. 23 (5), 881-895
- https://doi.org/10.1091/mbc.e11-08-0703
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.Keywords
This publication has 97 references indexed in Scilit:
- Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvationProceedings of the National Academy of Sciences of the United States of America, 2011
- Termination of autophagy and reformation of lysosomes regulated by mTORNature, 2010
- Distinct Subsets of Sit4 Holophosphatases Are Required for Inhibition of Saccharomyces cerevisiae Growth by Rapamycin and ZymocinEukaryotic Cell, 2009
- VAC14 nucleates a protein complex essential for the acute interconversion of PI3P and PI(3,5)P2 in yeast and mouseThe EMBO Journal, 2008
- TOR1 and TOR2 Have Distinct Locations in Live CellsEukaryotic Cell, 2008
- Atg18 Regulates Organelle Morphology and Fab1 Kinase Activity Independent of Its Membrane Recruitment by Phosphatidylinositol 3,5-BisphosphateMolecular Biology of the Cell, 2007
- Role of the V-ATPase in Regulation of the Vacuolar Fission–Fusion EquilibriumMolecular Biology of the Cell, 2007
- Phosphoinositide 5-Phosphatase Fig4p Is Required for both Acute Rise and Subsequent Fall in Stress-Induced Phosphatidylinositol 3,5-Bisphosphate LevelsEukaryotic Cell, 2006
- Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusionThe Journal of cell biology, 2005
- Targets for Cell Cycle Arrest by the Immunosuppressant Rapamycin in YeastScience, 1991