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...
Vac8p is a vacuolar membrane protein that is required for efficient vacuole inheritance and fusion, cytosol-to-vacuole targeting, and sporulation. By analogy to other armadillo domain proteins, including β-catenin and importin α, we hypothesize that Vac8p docks various factors at the vacuole membrane. Two-hybrid and copurfication assays demonstrated that Vac8p does form complexes with multiple binding partners, including Apg13p, Vab2p, and Nvj1p. Here we describe the surprising role of Vac8p-Nvj1p complexes in the formation of nucleus–vacuole (NV) junctions. Nvj1p is an integral membrane protein of the nuclear envelope and interacts with Vac8p in the cytosol through its C-terminal 40–60 amino acids (aa). Nvj1p green fluorescent protein (GFP) concentrated in small patches or rafts at sites of close contact between the nucleus and one or more vacuoles. Previously, we showed that Vac8p-GFP concentrated in intervacuole rafts, where is it likely to facilitate vacuole-vacuole fusion, and in “orphan” rafts at the edges of vacuole clusters. Orphan rafts of Vac8p red-sifted GFP (YFP) colocalize at sites of NV junctions with Nvj1p blue-sifted GFP (CFP). GFP-tagged nuclear pore complexes (NPCs) were excluded from NV junctions. In vac8-Δ cells, Nvj1p-GFP generally failed to concentrate into rafts and, instead, encircled the nucleus. NV junctions were absent in both nvj1-Δ andvac8-Δ cells. Overexpression of Nvj1p caused the profound proliferation of NV junctions. We conclude that Vac8p and Nvj1p are necessary components of a novel interorganelle junction apparatus.
OSH1 belongs to a seven-member gene family in yeast that is related to mammalian oxysterol-binding protein (OSBP). Here, we investigate the targeting of Osh1p to nucleus-vacuole (NV) junctions in Saccharomyces cerevisiae. NV junctions are interorganelle interfaces mediated by Nvj1p in the nuclear envelope and Vac8p on the vacuole membrane. Together, Nvj1p and Vac8p form Velcro-like patches through which teardrop-like portions of the nucleus are pinched off into the vacuolar lumen and degraded by a process termed piecemeal microautophagy of the nucleus (PMN). Osh1p is targeted to NV junctions proportional to NVJ1 expression through a physical association with Nvj1p. NV junctions per se are not required for this targeting because Osh1p colocalizes with Nvj1p in the absence of Vac8p. NV-junction-associated Osh1p is also a substrate for PMN degradation. Although OSH1 is not required for NV-junction formation or PMN, PMN is defective in cells lacking the yeast OSBP family (Osh1p to Osh7p). By contrast, the vesicular targeting of aminopeptidase I to the vacuole by macroautophagy is not dependent on the Osh protein family. We conclude the formation of nuclear PMN vesicles requires the overlapping activities of Osh1p and other Osh family members.
Previously published online as an AcKnowlEDGEmEntSWe wish to thank past and present lab members who have contributed their ideas and technical assistance to this project, especially Jonathan Millen who made comments on this manuscript, and to our many helpful and supportive colleagues in the autophagy research community. This study was supported by grants from the National Science Foundation and National Institutes of Health. AbStrActVarious modes of autophagy conspire to degrade virtually every compartment of the eukaryotic cell. In Saccharomyces cerevisiae, a process called "piecemeal microautophagy of the nucleus" (PMN) even pinches off and degrades nonessential portions of the nucleus. PMN is a constitutive process induced to high levels by starvation or rapamycin, an inhibitor of TOR kinase. PMN occurs at nucleus-vacuole (NV) junctions, which are Velcro-like patches formed by interactions between the vacuole membrane protein Vac8p and the outer-nuclear-membrane protein Nvj1p. In response to nutrient depletion, Nvj1p increasingly binds and sequesters two proteins with roles in lipid metabolism, Osh1p and Tsc13p. Tsc13p is required for the normal biogenesis of PMN vesicles. The sequestration of Osh1p by Nvj1p likely serves to negatively regulate the trafficking of tryptophan permease(s) to the plasma membrane. Thus, NV junctions and PMN orchestrate novel and sophisticated responses to nutrient limitation.
Plasma, a unique state of matter with properties similar to those of ionized gas, is an effective biological disinfectant. However, the mechanism through which nonthermal or "cold" plasma inactivates microbes on surfaces is poorly understood, due in part to challenges associated with processing and analyzing live cells on surfaces rather than in aqueous solution. Here, we employ membrane adsorption techniques to visualize the cellular effects of plasma on representative clinical isolates of drug-resistant microbes. Through direct fluorescent imaging, we demonstrate that plasma rapidly inactivates planktonic cultures, with >5 log 10 kill in 30 s by damaging the cell surface in a time-dependent manner, resulting in a loss of membrane integrity, leakage of intracellular components (nucleic acid, protein, ATP), and ultimately focal dissolution of the cell surface with longer exposure time. This occurred with similar kinetic rates among methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Candida albicans. We observed no correlative evidence that plasma induced widespread genomic damage or oxidative protein modification prior to the onset of membrane damage. Consistent with the notion that plasma is superficial, plasma-mediated sterilization was dramatically reduced when microbial cells were enveloped in aqueous buffer prior to treatment. These results support the use of nonthermal plasmas for disinfecting multidrug-resistant microbes in environmental settings and substantiate ongoing clinical applications for plasma devices. P lasmas are ionized gases that exhibit a plethora of applied temperature and physical properties. In biomedical applications, plasmas are supported by an electric field such that electrons receive external energy more rapidly than surrounding ions (5). Plasmas generate thermal energy (heat) when heavy particle temperatures equilibrate with electron temperature but are considered "nonthermal" when the cooling of ions and uncharged molecules is more effective than the energy transfer from electrons to gas (5). Nonthermal or "cold" plasmas produce a variety of shortlived and long-lived reactive components, including charged particles and UV radiation, without significantly raising temperature (25). Nonthermal plasmas (NTPs) are applied extensively in materials science to modify the properties of carbon-based materials but have also shown promise for applications in biology and med-
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