The Saccharomyces cerevisiae vacuole serves as the main storage compartment for essential amino acids, nutrients and ions, functions in the regulated turnover of macromolecules, and is required for sporulation and osmotic homeostasis. Rapid changes in size, shape, and number are critical to ensure an immediate response to dramatic fluctuations in nutrient concentrations and extracellular osmolarity. However, even under relatively static external conditions, the vacuole is in a constant state of regulated flux. The multiple membrane transport pathways that converge on the vacuole are kept in balance to maintain the the proper function and morphology of the vacuole.
We have characterized the Golgi protein Mon2 in an effort to elucidate the connections between homeostasis of this organelle and various functions of the vacuole. The highly fragmented vacuoles of the mon2Δ mutant underscore the requirement for a stable Golgi appartus in vacuole biogenesis. Furthermore, our finding that Mon2p is required for cytoplasm-to-vacuole transport indicates that the Golgi, aside from being a critical way station along membrane and protein transport pathways culminating at the vacuole, is likely also a membrane source for autophagic process(es).
Further downstream in anterograde membrane traffic, endosomal maturation defects result in various functional and structural anomalies of the vacuole. We show here that bacterial proteins from Legionella pneumophila, when overexpressed, can inhibit multivesicular body formation, leading to missorting of the hydrolase Carboxypeptidase Y and to the formation of an aberrant membrane compartment adjacent to the vacuole. A genetic screen was used to identify such proteins, all of which were subsequently found to be released into the host cytosol upon Legionella infection.
At the vacuolar compartment itself, the phosphoinositide PtdIns(3,5) P2 plays a key role in regulating morphology. Our findings indicate that the first bona fide PtdIns(3,5)P2 effector, Atg18, is both necessary and sufficient for the fragmentation of the vacuole observed when levels of this phosphoinositide increase. Moreover, Atg18 is a potent inhibitor of the Fab1 kinase, potentially modulating interaction with primary upstream activator, Vac7. Finally, Atg18 also interacts with Vac17p, indicating a possible function in retrograde membrane transport and/or vacuole inheritance.