CLIP and cohibin separate rDNA from nucleolar proteins destined for degradation by nucleophagy.
06 Aug 2018-Journal of Cell Biology (The Rockefeller University Press)-Vol. 217, Iss: 8, pp 2675-2690
TL;DR: It is shown that TORC1 inactivation promotes relocalization of nucleolar proteins and rDNA to different sites, and that rDNA–nucleolar protein separation is important for the nucleophagic degradation ofucleolar proteins.
Abstract: Nutrient starvation or inactivation of target of rapamycin complex 1 (TORC1) in budding yeast induces nucleophagy, a selective autophagy process that preferentially degrades nucleolar components. DNA, including ribosomal DNA (rDNA), is not degraded by nucleophagy, even though rDNA is embedded in the nucleolus. Here, we show that TORC1 inactivation promotes relocalization of nucleolar proteins and rDNA to different sites. Nucleolar proteins move to sites proximal to the nuclear–vacuolar junction (NVJ), where micronucleophagy (or piecemeal microautophagy of the nucleus) occurs, whereas rDNA dissociates from nucleolar proteins and moves to sites distal to NVJs. CLIP and cohibin, which tether rDNA to the inner nuclear membrane, were required for repositioning of nucleolar proteins and rDNA, as well as effective nucleophagic degradation of the nucleolar proteins. Furthermore, micronucleophagy itself was necessary for the repositioning of rDNA and nucleolar proteins. However, rDNA escaped from nucleophagic degradation in CLIP- or cohibin-deficient cells. This study reveals that rDNA–nucleolar protein separation is important for the nucleophagic degradation of nucleolar proteins.
Citations
More filters
TL;DR: The data suggest that perturbation to the nuclear envelope barrier would lead to local nuclear membrane remodeling to promote membrane sealing, and have implications for disease mechanisms linked to NPC assembly and nuclear envelope integrity.
Abstract: The integrity of the nuclear membranes coupled to the selective barrier of nuclear pore complexes (NPCs) are essential for the segregation of nucleoplasm and cytoplasm. Mechanical membrane disruption or perturbation to NPC assembly triggers an ESCRT-dependent surveillance system that seals nuclear pores: how these pores are sensed and sealed is ill defined. Using a budding yeast model, we show that the ESCRT Chm7 and the integral inner nuclear membrane (INM) protein Heh1 are spatially segregated by nuclear transport, with Chm7 being actively exported by Xpo1/Crm1. Thus, the exposure of the INM triggers surveillance with Heh1 locally activating Chm7. Sites of Chm7 hyperactivation show fenestrated sheets at the INM and potential membrane delivery at sites of nuclear envelope herniation. Our data suggest that perturbation to the nuclear envelope barrier would lead to local nuclear membrane remodeling to promote membrane sealing. Our findings have implications for disease mechanisms linked to NPC assembly and nuclear envelope integrity.
84 citations
TL;DR: Recent discoveries in nuclear recycling, namely nucleophagy in physiology in yeast and nucleophagic events that occur in pathological conditions in mammals are presented.
Abstract: Nuclear abnormalities are prominent in degenerative disease and progeria syndromes. Selective autophagy of organelles is instrumental in maintaining cell homeostasis and prevention of premature ageing. Although the nucleus is the control centre of the cell by safeguarding our genetic material and controlling gene expression, little is known in relation to nuclear autophagy. Here we present recent discoveries in nuclear recycling, namely nucleophagy in physiology in yeast and nucleophagic events that occur in pathological conditions in mammals. The selective nature of degrading nuclear envelope components, DNA, RNA and nucleoli is highlighted. Potential effects of perturbed nucleophagy in senescence and longevity are examined. Moreover, the open questions that remain to be explored are discussed concerning the conditions, receptors and substrates in homeostatic nucleophagy.
73 citations
TL;DR: It is shown that, after nitrogen starvation and genetic interference with the architecture of nuclear pore complexes, nucleoporins are degraded by autophagy, constituting a quality-control step at the nuclear envelope.
Abstract: Nuclear pore complexes (NPCs) are very large proteinaceous assemblies that consist of more than 500 individual proteins1,2. NPCs are essential for nucleocytoplasmic transport of different cellular components, and disruption of the integrity of NPCs has been linked to aging, cancer and neurodegenerative diseases3-7. However, the mechanism by which membrane-embedded NPCs are turned over is currently unknown. Here we show that, after nitrogen starvation or genetic interference with the architecture of NPCs, nucleoporins are rapidly degraded in the budding yeast Saccharomyces cerevisiae. We demonstrate that NPC turnover involves vacuolar proteases and the core autophagy machinery. Autophagic degradation is mediated by the cytoplasmically exposed Nup159, which serves as intrinsic cargo receptor and directly binds to the autophagy marker protein Atg8. Autophagic degradation of NPCs is therefore inducible, enabling the removal of individual NPCs from the nuclear envelope.
70 citations
TL;DR: A strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments is developed and their composition is analyzed by using quantitative mass spectrometry to provide a comprehensive picture of heterochromeatin-associated proteins and suggest a role for specific nucleoporins in heterochromaatin function.
63 citations
TL;DR: It is shown that Lro1, an acyltransferase that generates TGs from phospholipid-derived FAs in yeast, relocates from the endoplasmic reticulum to a subdomain of the inner nuclear membrane, and its compartmentalization is critical for nuclear integrity.
48 citations
References
More filters
TL;DR: The results on the apg mutants suggest that autophagy via autophagic bodies is indispensable for protein degradation in the vacuoles under starvation conditions, and that at least 15 APG genes are involved inAutophagy in yeast.
1,653 citations
"CLIP and cohibin separate rDNA from..." refers background in this paper
...The CLIP–cohibin axis is required for survival during nutrient starvation Loss of the core autophagy component Atg1 rapidly reduced cell survivability during nitrogen starvation (Tsukada and Ohsumi, 1993), indicating that autophagy induction after nutrient starvation is critical for cell survival....
[...]
TL;DR: The discovery of autophagy in yeast and the genetic tractability of this organism have allowed us to identify genes that are responsible for this process, which has led to the explosive growth of this research field seen today.
Abstract: Autophagy is a fundamental function of eukaryotic cells and is well conserved from yeast to humans. The most remarkable feature of autophagy is the synthesis of double membrane-bound compartments that sequester materials to be degraded in lytic compartments, a process that seems to be mechanistically distinct from conventional membrane traffic. The discovery of autophagy in yeast and the genetic tractability of this organism have allowed us to identify genes that are responsible for this process, which has led to the explosive growth of this research field seen today. Analyses of autophagy-related (Atg) proteins have unveiled dynamic and diverse aspects of mechanisms that underlie membrane formation during autophagy.
1,511 citations
"CLIP and cohibin separate rDNA from..." refers background in this paper
...Macroautophagy degrades cytoplasmic components and organelles in lysosomes/vacuoles, which is a conserved system from yeast to mammalian cells (Nakatogawa et al., 2009; Reggiori and Klionsky, 2013)....
[...]
TL;DR: Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle.
Abstract: For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.
1,151 citations
"CLIP and cohibin separate rDNA from..." refers background in this paper
...It was indeed the case: the repositioning of nucleolar proteins and rDNA already occurred after 1 h of TORC1 inactivation (as mentioned above), although autophagy is not significantly induced after 1 h of nutrient starvation and TORC1 inactivation (Takeshige et al., 1992)....
[...]
...It was indeed the case: the repositioning of nucleolar proteins and rDNA already occurred after 1 h of TORC1 inactivation (as mentioned above), although autophagy is not significantly induced after 1 h of nutrient starvation and TORC1 inactivation (Takeshige et al., 1992)....
[...]
TL;DR: In this method, yeast cells are subjected to mild alkali treatment and then boiled in a standard electrophoresis loading buffer, which yields virtually complete extraction independently of the strain, growth conditions and protein molecular weight.
Abstract: The methods currently used for protein extraction from yeast are either laborious or insufficiently reliable. Here I report a method for protein extraction for electrophoretic analysis that is both easy and reliable. In this method, yeast cells are subjected to mild alkali treatment and then boiled in a standard electrophoresis loading buffer. The method was tested for different strains of Saccharomyces cerevisiae and for yeast Hansenula polymorpha DL-1. It yields virtually complete extraction independently of the strain, growth conditions and protein molecular weight and allows working with small amounts of yeast cells grown on agar plates. Copyright © 2000 John Wiley & Sons, Ltd.
905 citations
"CLIP and cohibin separate rDNA from..." refers methods in this paper
...Proteins were extracted using a postalkaline extraction method in accordance with a previous study (Kushnirov, 2000)....
[...]
TL;DR: The yeast Saccharomyces cerevisiae has played a prominent role in both the discovery of TOR and the elucidation of its function, and the TOR signaling network in S. cerevisia is reviewed.
Abstract: TOR (Target Of Rapamycin) is a highly conserved protein kinase that is important in both fundamental and clinical biology. In fundamental biology, TOR is a nutrient-sensitive, central controller of cell growth and aging. In clinical biology, TOR is implicated in many diseases and is the target of the drug rapamycin used in three different therapeutic areas. The yeast Saccharomyces cerevisiae has played a prominent role in both the discovery of TOR and the elucidation of its function. Here we review the TOR signaling network in S. cerevisiae.
794 citations
"CLIP and cohibin separate rDNA from..." refers background in this paper
...Nitrogen starvation causes TORC1 inactivation and is a natural trigger of autophagy induction (Loewith and Hall, 2011)....
[...]