In mammalian liver, proteolysis is regulated by the cellular hydration state in a microtubule- and p38(MAPK) (p38 mitogen-activated protein kinase)-dependent fashion. Osmosensing in liver cells towards proteolysis is achieved by activation of integrin receptors. The yeast orthologue of p38(MAPK) is Hog1 (high-osmolarity glycerol 1), which is involved in the hyperosmotic-response pathway. Since it is not known whether starvation-induced autophagy in yeast is osmosensitive and whether Hog1 is involved in this process, we performed fluorescence microscopy experiments. The hog1Delta cells exhibited a visible decrease of autophagy in hypo-osmotic and hyperosmotic nitrogen-starvation medium as compared with normo-osmolarity, as determined by GFP (green fluorescent protein)-Atg8 (autophagy-related 8) fluorescence. Western blot analysis of GFP-Atg8 degradation showed that WT (wild-type) cells maintained a stable autophagic activity over a broad osmolarity range, whereas hog1Delta cells showed an impaired autophagic actitivity during hypo- and hyper-osmotic stress. In [3H]leucine-pre-labelled yeast cells, the proteolysis rate was osmodependent only in hog1Delta cells. Neither maturation of pro-aminopeptidase I nor vitality was affected by osmotic stress in either yeast strain. In contrast, rapamycin-dependent autophagy, as measured by degradation of GFP-Atg8, did not significantly respond to hypo-osmotic or hyperosmotic stress in hog1Delta or WT cells. We conclude that Hog1 plays a role in the stabilization machinery of nitrogen-deprivation-induced autophagy in yeast cells during ambient osmolarity changes. This could be an analogy to the p38(MAPK) pathway in mammalian liver, where osmosensing towards p38(MAPK) is required for autophagy regulation by hypo-osmotic or amino-acid-induced cell swelling. A phenotypic difference is observed in rapamycin-induced autophagy, which does not seem to respond to extracellular osmolarity changes in hog1Delta cells.

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Journal Article | Research Support, Non-U.S. Gov't

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Prick T, Thumm M, Köhrer K, Häussinger D, Vom Dahl S

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