Target of rapamycin signaling mediates vacuolar fragmentation

Abstract

In eukaryotic cells, cellular homeostasis requires that different organelles respond to intracellular as well as environmental signals and modulate their behavior as conditions demand. Understanding the molecular mechanisms required for these changes remains an outstanding goal. One such organelle is the lysosome/vacuole, which undergoes alterations in size and number in response to environmental and physiological stimuli. Changes in the morphology of this organelle are mediated in part by the equilibrium between fusion and fission processes. While the fusion of the yeast vacuole has been studied intensively, the regulation of vacuolar fission remains poorly characterized by comparison. In recent years, a number of studies have incorporated genome-wide visual screens and high-throughput microscopy to identify factors required for vacuolar fission in response to diverse cellular insults, including hyperosmotic and endoplasmic reticulum stress. Available evidence now demonstrates that the rapamycin-sensitive TOR network, a master regulator of cell growth, is required for vacuolar fragmentation in response to stress. Importantly, many of the genes identified in these studies provide new insights into potential links between the vacuolar fission machinery and TOR signaling. Together these advances both extend our understanding of the regulation of vacuolar fragmentation in yeast as well as underscore the role of analogous events in mammalian cells.

Keywords: ER stress; TORC1; Vacuolar fission.

Fig. 1

Vacuolar morphology is responsive to cellular stress. (A) Model of changes in vacuolar morphology mediated by an equilibrium between fission and fusion processes. Under hypo-osmotic conditions, rapamycin treatment, or nutrient limitation, vacuoles are one large organelle. In response to Hyper-osmotic, ER, and Lactic acid stress, vacuoles fragment into multiple, smaller organelles. (B) Visualization of vacuolar morphology using FM4-64. WT (W303 ) cells were treated overnight with FM4-64, then live cells were imaged following treatment with DMSO (Normal), 1μg/mL Tm (ER Stress), and 200nM Rapamycin (Nutrient Limitation)

Fig. 2

Model for ER stress-induced vacuolar fragmentation. ER stress and TORC1 are likely to act in parallel to influence vacuolar morphology. While components downstream of ER stress remain to be identified, both TORC1 effector branches mediated by Tap42/Sit4 and Sch9 are known to be required for vacuolar fragmentation incited by ER stress. Additionally, Vph2, an assembly factor for the V-ATPase, as well as the V-ATPase itself are also required for ER stress-induced vacuolar fragmentation. Interestingly, the V-ATPase is required for vacuolar fission in response to ER stress; however, this role appears to be distinct from its role in maintaining acidification of the vacuolar compartment. At present, the potential role of Sch9 and/or Tap42/Sit4 in Vph2 regulation remains speculative, and is based upon TORC1-dependent changes in Vph2 localization in response to ER stress in a TORC1 dependent manner

Fig. 3

Genome-wide screen for genes involved in ER stress-induced vacuolar fragmentation. Approximately 5000 strains contained in the yeast haploid deletion collection were grown overnight (16h) in YPD medium containing 1μM FM4-64. Cells were diluted 1:25 in fresh medium for 3h to allow for logarithmic phase growth, after which ER stress was induced by adding YPD medium containing 1μg/mL Tm. Treated cells were transferred to glass bottomed microscopy plates treated with concanavalin A and imaged using the CellVoyager CV1000 confocal system, using a 60Å~ water immersion objective. Deletion strains with 50% or more of cells within the population containing a defect in vacuolar fragmentation (non-fragmented) were considered to be candidate hits. Potential hits from the initial pass were re-arrayed using a RoToR robot to form a new library of candidate hits that was assayed twice more as described above and, in addition, following treatment with 25μM DTT. 315 strains that contained cells with >50% non-fragmented vacuoles were considered hits and manually grouped according to their cellular function.