Final Report Summary - ORGANELL (Organelle homeostasis: How are membrane fission and fusion machineries coordinated to regulate size and copy number of a lysosomal compartment?) We completed a genome-wide screen for genes affecting vacuole fission in vivo and reconstituted vacuole fission in a cell-free system with purified vacuoles. The in vitro reaction could be quantified by processing of microscopic images. We identified an ordered sequence of intermediate stages of vacuole fission and assigned distinct protein and lipid requirements to them. These assays, combined with the results from genetic screening, allowed us to identify PROPPINs as a novel family of membrane fission proteins that are present in all eukaryotes. A yeast PROPPIN was purified and its membrane tubulation and membrane fission activity could be demonstrated with giant uni-lamellar liposomes. Structure-function studies provided us insight into the fission mechanism of PROPPINs and allowed us to develop a hypothesis on their mode of action. Complementary studies in mammalian cells indicate that PROPPINs have strong impact on cargo transport between lysosomes and various endosomes and on the size and number of these compartments. The combined evidence indicates that PROPPINs act on multiple organelles of the endo-lysosomal system and that they represent a novel family of eukaryotic membrane fission proteins. Our studies on the control of vacuole size and number by organelle required means to manipulate the gross content of vacuoles in order to address the question whether volume and copy number of the organelle envelope are regulated in response to the amount of content to be enclosed. In developing methods to manipulate the gross content of vacuoles we made a fundamental new discovery for metabolic regulation in eukaryotes. We had to explore the regulation of the vacuolar polyphosphate polymerase VTC, which is responsible for synthesizing inorganic polyphosphate, the dominant lumenal compound that makes up more than 50% of the dry weight of a vacuole. We discovered that VTC is regulated by a domain of hitherto unknown function, SPX. We determined the structure of SPX, identified inositol pyrophosphates as their physiological ligands and established a novel signal transduction pathway that communicates cytosolic phosphate levels via inositol pyrophosphates to a multitude of proteins that import, export or store phosphate, or regulate phosphate-dependent gene transcription. All these proteins carry SPX domains and their coordinated regulation guarantees the homeostasis of inorganic phosphate in the cytosol. This has great impact on all nucleotide hydrolysing reactions.These studies also gave us the tools to demonstrate that the loading status of vacuoles is communicated from their lumen to the organelle surface. Here, it controls the activation of the membrane fusion machinery by regulating the binding of NSF/Sec18 to vacuolar SNARE complexes. Therefore, we postulated that vacuolar content is signalled from the organelle lumen to its surface, where it can increase storage space by inducing fusion of several organelle copies into a single organelle of increase volume but identical surface.
