Piecemeal microautophagy of the nucleus: genetic and morphological traits.

Nucleus-vacuole (NV) junctions are formed in Saccharomyces cerevisiae through interactions between Vac8 in the vacuole membrane and Nvj1 in the perinuclear ER. Upon starvation, vesicles containing part of the nucleus emanate from these contact sites and finally pinch off into invaginations of the vacuole. Due to its morphological similarity to microautophagy this process had been termed "piecemeal microautophagy of the nucleus" (PMN). We recently discovered that a number of ATG genes required for macroautophagy and micropexophagy are also required for PMN and accordingly named it micronucleophagy. Therefore, PMN represents a novel model system to investigate the functions of the highly conserved but poorly understood core autophagic apparatus. We here extend the morphological analysis of PMN using immunogold and freeze fracture electron microscopy.

Measuring piecemeal microautophagy of the nucleus in Saccharomyces cerevisiae.

Piecemeal microautophagy of the nucleus (PMN) selectively removes and degrades small fragments of the Saccharomyces cerevisiae nucleus. Inter-organelle contact sites called nucleus-vacuole (NV) junctions determine the selectivity of PMN by establishing a platform for the biogenesis of PMN blebs and vesicles. PMN structures can be observed by fluorescence microscopy using GFP-tagged reporters; however, this approach is best supported with quantitative immunoblot assays of PMN-specific cargo degradation. Together, these assays should facilitate the further study of this fascinating but poorly understood autophagic process in different genetic backgrounds, physiological states, and environmental conditions.

Measuring macroautophagy in S. cerevisiae: autophagic body accumulation and total protein turnover.

Macroautophagy has been implicated in various physiological functions and severe human diseases. Accordingly, there is a high interest in determining macroautophagy both qualitatively and quantitatively. In this chapter we discuss how macroautophagy can be followed morphologically in the yeast Saccharomyces cerevisiae using light microscopy. To quantitatively measure macroautophagy, we further present two protocols for the determination of total protein turnover.

Deletion of HOG1 leads to Osmosensitivity in starvation-induced, but not rapamycin-dependent Atg8 degradation and proteolysis: further evidence for different regulatory mechanisms in yeast autophagy.

The mechanisms of regulation of autophagy are still obscure. In mammalian liver, starvation-induced autophagic proteolysis is regulated by the cellular hydration state in a microtubule- and p38(MAPK)-dependent way. Recent work shows that in yeast, loss of Hog1, the yeast orthologue of p38(MAPK), leads to osmosensitivity of starvation-induced autophagy (Prick et al., Biochem J 2006; 394:153-161), pointing to an evolutionarily conserved mechanism. In this addendum further experiments from hog1Delta yeast cells are shown, which support the hypothesis that starvation- and rapamycin-induced autophagy processes differ in their susceptibility to osmotic stress. The potential mechanisms are discussed.

In yeast, loss of Hog1 leads to osmosensitivity of autophagy.

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.