Piecemeal Microautophagy of Nucleus inSaccharomyces cerevisiae

Abstract

Nucleus-vacuole (NV) junctions in Saccharomyces cerevisiae are formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the nuclear envelope. Herein, we report that NV junctions in yeast promote piecemeal microautophagy of the nucleus (PMN). During PMN, teardrop-like blebs are pinched from the nucleus, released into the vacuole lumen, and degraded by soluble hydrolases. PMN occurs in rapidly dividing cells but is induced to higher levels by carbon and nitrogen starvation and is under the control of the Tor kinase nutrient-sensing pathway. Confocal and biochemical assays demonstrate that Nvj1p is degraded in a PMN-dependent manner. PMN occurs normally in apg7-⌬ cells and is, therefore, not dependent on macroautophagy. Transmission electron microscopy reveals that portions of the granular nucleolus are often sequestered into PMN structures. These results introduce a novel mode of selective microautophagy that targets nonessential components of the yeast nucleus for degradation and recycling in the vacuole. INTRODUCTIONAutophagy functions in dividing cells to recycle the cytoplasm and is essential for cell viability during extended periods of starvation (Klionsky and Ohsumi, 1999). Autophagy in yeast and mammals occurs by various modes, including morphologically distinct macro-and microautophagic pathways. Macroautophagy in Saccharomyces cerevisiae is induced by starvation and involves the formation of double membrane autophagosomes around bulk cytoplasm and organelles (Takeshige et al., 1992;Baba et al., 1994). Vesicular targeting factors mediate the fusion of the outer autophagosomal membrane with the vacuole (Darsow et al., 1997;Sato et al., 1998), and an autophagic body is subsequently released into the vacuole lumen (Baba et al., 1994) where it is degraded by acid hydrolases (Jones et al., 1997). Most vacuolar hydrolases are synthesized as inactive proenzymes, which are activated in the vacuole by Pep4p and Prb1p proteinases. Thus, autophagic bodies accumulate in the vacuoles of pep4 or prb1 mutant cells (Takeshige et al., 1992;Woolford et al., 1993;Baba et al., 1994;Jones et al., 1997) due to their slower degradation rates (Jones et al., 1982;Zubenko et al., 1983).Many of the factors necessary for the formation of autophagosomes are used in the cytosol-to-vacuole targeting (Cvt) of proaminopeptidase I to the vacuole lumen (Scott et al., 1996;Teter and Klionsky, 2000). APG/AUT/CVT genes, which are required for the formation of Cvt vesicles and their conversion into larger autophagosomes (Abeliovich et al., 2000;Kim et al., 2001a), also comprise components of a novel system of ubiquitin-like conjugation reactions (Klionsky and Ohsumi, 1999). Common to these reactions is Apg7p, a conserved E1-like enzyme (Mizushima et al., 1998a,b) that is required both for the conjugation of Apg12p to Apg5p and of Aut7p/Apg8p to phosphatidylethanolamine (Ichimura et al., 2000). Recently, it was shown that some Apg proteins, including Apg5p and Aut7p/Apg8p, are required for early steps in the fo...

Cellular microcompartments constitute general suborganellar functional units in cells

“…In yeast, contact sites between the nucleus and vacuole, termed nucleus-vacuole junctions (NVJ), form by the association of the vacuolar protein Vac8 and the ER-resident protein Nvj1 ( Figure 3C). NVJ formation is necessary for piecemeal microautopaghy of the nucleus (PMN), which can be induced upon carbon or nitrogen starvation (Roberts et al , 2003 ). The sterol-binding protein Osh1 and fatty acid elongase Tsc13 localize to the NVJ.…”

Selective and Non-Selective Autophagic Degradation of Mitochondria in Yeast

“…[9][10][11] A degradation of portions of the nucleus, termed Piecemeal Microautophagy of the Nucleus (PMN), has been reported in the yeast Saccharomyces cerevisiae submitted to nitrogen starvation. 12 PMN involves a direct contact between the vacuole and the nucleus, the formation of junctions, and the protrusion of parts of the nucleus inside the vacuole, forming vesicles that are later degraded. The formation of these junctions has been shown to involve a protein-protein interaction between Nvj1p 13 on the nuclear envelope and Vac8p on the vacuolar membrane.…”

“…The formation of these junctions has been shown to involve a protein-protein interaction between Nvj1p 13 on the nuclear envelope and Vac8p on the vacuolar membrane. 12 In addition to their well-established functions in oxidative phosphorylation and numerous metabolic pathways, the role played by mitochondrial homeostasis in cellular ageing and programmed cell death, has, in recent years, provided new investigative pathways to major physiological and pathological issues. 14 Indeed, mitochondria are both a major source and the main target of reactive oxygen species that are the principal cause of the alteration of biological structures.…”

Impairing the bioenergetic status and the biogenesis of mitochondria triggers mitophagy in yeast

“…Diverging descriptions of microautophagy have been documented in S. cerevisiae, concerning its dependence on the Apg/Aut pathway 12 and TOR. 13 In higher eukaryotes, complete autophagy is generally acknowledged as a PCD. Recent studies report that type II PCD (autophagy) could substitute for defective type I PCD (apoptosis), suggesting (i) that autophagy could be physiologically triggered by other means besides nutritional stress, and (ii) that autophagy could be an alternative to apoptosis.…”