A Trio of Ubiquitin Ligases Sequentially Drive Ubiquitylation and Autophagic Degradation of Dysfunctional Yeast Proteasomes

Mapping Intimacies ◽

10.1101/2021.05.12.443936 ◽

2021 ◽

Author(s):

Richard S. Marshall ◽

Richard D. Vierstra

Keyword(s):

Nuclear Export ◽

Ubiquitin Ligases ◽

Misfolded Proteins ◽

Autophagic Degradation ◽

Multiple Levels ◽

Regulatory Particle

As central effectors of ubiquitin (Ub)-mediated proteolysis, proteasomes are regulated at multiple levels, including degradation of unwanted or dysfunctional particles via autophagy (termed proteaphagy). In yeast, inactive proteasomes are exported from the nucleus, sequestered into cytoplasmic aggresomes via the Hsp42 chaperone, extensively ubiquitylated, and then tethered to the expanding phagophore by the autophagy receptor Cue5. Here, we demonstrate the need for ubiquitylation driven by the trio of Ub ligases (E3s) San1, Rsp5 and Hul5, which, together with their corresponding E2s, work sequentially to promote nuclear export and Cue5 recognition. Whereas San1 functions prior to nuclear export, Rsp5 and Hul5 likely decorate aggresome-localized proteasomes in concert. Ultimately, topologically complex Ub chain(s) containing both K48 and K63 Ub-Ub linkages are assembled, mainly on the regulatory particle, to generate autophagy-competent substrates. As San1, Rsp5, Hul5, Hsp42, and Cue5 also participate in general proteostasis, proteaphagy likely engages an essential mechanism for eliminating inactive/misfolded proteins.

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Exposed hydrophobicity is a key determinant of nuclear quality control degradation

Molecular Biology of the Cell ◽

10.1091/mbc.e11-03-0256 ◽

2011 ◽

Vol 22 (13) ◽

pp. 2384-2395 ◽

Cited By ~ 62

Author(s):

Eric K. Fredrickson ◽

Joel C. Rosenbaum ◽

Melissa N. Locke ◽

Thomas I. Milac ◽

Richard G. Gardner

Keyword(s):

Quality Control ◽

Ubiquitin Ligase ◽

Protein Quality ◽

Ubiquitin Ligases ◽

Protective Role ◽

Nuclear Proteins ◽

Misfolded Proteins ◽

Structural Abnormality ◽

Cellular Toxicity ◽

Hydrophobic Residues

Protein quality control (PQC) degradation protects the cell by preventing the toxic accumulation of misfolded proteins. In eukaryotes, PQC degradation is primarily achieved by ubiquitin ligases that attach ubiquitin to misfolded proteins for proteasome degradation. To function effectively, PQC ubiquitin ligases must distinguish misfolded proteins from their normal counterparts by recognizing an attribute of structural abnormality commonly shared among misfolded proteins. However, the nature of the structurally abnormal feature recognized by most PQC ubiquitin ligases is unknown. Here we demonstrate that the yeast nuclear PQC ubiquitin ligase San1 recognizes exposed hydrophobicity in its substrates. San1 recognition is triggered by exposure of as few as five contiguous hydrophobic residues, which defines the minimum window of hydrophobicity required for San1 targeting. We also find that the exposed hydrophobicity recognized by San1 can cause aggregation and cellular toxicity, underscoring the fundamental protective role for San1-mediated PQC degradation of misfolded nuclear proteins.

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gp78 functions downstream of Hrd1 to promote degradation of misfolded proteins of the endoplasmic reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.e15-06-0354 ◽

2015 ◽

Vol 26 (24) ◽

pp. 4438-4450 ◽

Cited By ~ 34

Author(s):

Ting Zhang ◽

Yue Xu ◽

Yanfen Liu ◽

Yihong Ye

Keyword(s):

Endoplasmic Reticulum ◽

Ubiquitin Ligase ◽

Ubiquitin Ligases ◽

Misfolded Proteins ◽

Ubiquitin Ligase Complex ◽

Evolutionarily Conserved ◽

Membrane Bound ◽

Complex Forms ◽

Functional Relevance ◽

Chaperone Complex

Eukaryotic cells eliminate misfolded proteins from the endoplasmic reticulum (ER) via a conserved process termed ER-associated degradation (ERAD). Central regulators of the ERAD system are membrane-bound ubiquitin ligases, which are thought to channel misfolded proteins through the ER membrane during retrotranslocation. Hrd1 and gp78 are mammalian ubiquitin ligases homologous to Hrd1p, an ubiquitin ligase essential for ERAD in Saccharomyces cerevisiae. However, the functional relevance of these proteins to Hrd1p is unclear. In this paper, we characterize the gp78-containing ubiquitin ligase complex and define its functional interplay with Hrd1 using biochemical and recently developed CRISPR-based genetic tools. Our data show that transient inactivation of the gp78 complex by short hairpin RNA–mediated gene silencing causes significant stabilization of both luminal and membrane ERAD substrates, but unlike Hrd1, which plays an essential role in retrotranslocation and ubiquitination of these ERAD substrates, knockdown of gp78 does not affect either of these processes. Instead, gp78 appears to act downstream of Hrd1 to promote ERAD via cooperation with the BAG6 chaperone complex. We conclude that the Hrd1 complex forms an essential retrotranslocation module that is evolutionarily conserved, but the mammalian ERAD system uses additional ubiquitin ligases to assist Hrd1 during retrotranslocation.

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How Is the Fidelity of Proteins Ensured in Terms of Both Quality and Quantity at the Endoplasmic Reticulum? Mechanistic Insights into E3 Ubiquitin Ligases

International Journal of Molecular Sciences ◽

10.3390/ijms22042078 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2078

Author(s):

Ji An Kang ◽

Young Joo Jeon

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Molecular Mechanisms ◽

Protein Quality ◽

Protein Quality Control ◽

Ubiquitin Ligases ◽

Ubiquitin Proteasome System ◽

Misfolded Proteins ◽

E3 Ubiquitin Ligases ◽

Quantity Control

The endoplasmic reticulum (ER) is an interconnected organelle that plays fundamental roles in the biosynthesis, folding, stabilization, maturation, and trafficking of secretory and transmembrane proteins. It is the largest organelle and critically modulates nearly all aspects of life. Therefore, in the endoplasmic reticulum, an enormous investment of resources, including chaperones and protein folding facilitators, is dedicated to adequate protein maturation and delivery to final destinations. Unfortunately, the folding and assembly of proteins can be quite error-prone, which leads to the generation of misfolded proteins. Notably, protein homeostasis, referred to as proteostasis, is constantly exposed to danger by flows of misfolded proteins and subsequent protein aggregates. To maintain proteostasis, the ER triages and eliminates terminally misfolded proteins by delivering substrates to the ubiquitin–proteasome system (UPS) or to the lysosome, which is termed ER-associated degradation (ERAD) or ER-phagy, respectively. ERAD not only eliminates misfolded or unassembled proteins via protein quality control but also fine-tunes correctly folded proteins via protein quantity control. Intriguingly, the diversity and distinctive nature of E3 ubiquitin ligases determine efficiency, complexity, and specificity of ubiquitination during ERAD. ER-phagy utilizes the core autophagy machinery and eliminates ERAD-resistant misfolded proteins. Here, we conceptually outline not only ubiquitination machinery but also catalytic mechanisms of E3 ubiquitin ligases. Further, we discuss the mechanistic insights into E3 ubiquitin ligases involved in the two guardian pathways in the ER, ERAD and ER-phagy. Finally, we provide the molecular mechanisms by which ERAD and ER-phagy conduct not only protein quality control but also protein quantity control to ensure proteostasis and subsequent organismal homeostasis.

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Proteasome storage granules protect proteasomes from autophagic degradation upon carbon starvation

eLife ◽

10.7554/elife.34532 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 40

Author(s):

Richard S Marshall ◽

Richard D Vierstra

Keyword(s):

Nitrogen Starvation ◽

Carbon Starvation ◽

26S Proteasome ◽

Energy Status ◽

Cellular Energy ◽

The Core ◽

Evolutionarily Conserved ◽

Autophagic Degradation ◽

Storage Granules ◽

Multiple Levels

26S proteasome abundance is tightly regulated at multiple levels, including the elimination of excess or inactive particles by autophagy. In yeast, this proteaphagy occurs upon nitrogen starvation but not carbon starvation, which instead stimulates the rapid sequestration of proteasomes into cytoplasmic puncta termed proteasome storage granules (PSGs). Here, we show that PSGs help protect proteasomes from autophagic degradation. Both the core protease and regulatory particle sub-complexes are sequestered separately into PSGs via pathways dependent on the accessory proteins Blm10 and Spg5, respectively. Modulating PSG formation, either by perturbing cellular energy status or pH, or by genetically eliminating factors required for granule assembly, not only influences the rate of proteasome degradation, but also impacts cell viability upon recovery from carbon starvation. PSG formation and concomitant protection against proteaphagy also occurs in Arabidopsis, suggesting that PSGs represent an evolutionarily conserved cache of proteasomes that can be rapidly re-mobilized based on energy availability.

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It's not just a phase; ubiquitination in cytosolic protein quality control

Biochemical Society Transactions ◽

10.1042/bst20200694 ◽

2021 ◽

Author(s):

Heather A. Baker ◽

Jonathan P. Bernardini

Keyword(s):

Protein Misfolding ◽

Protein Quality ◽

Genetic Diseases ◽

Ubiquitin Ligases ◽

Ubiquitin Proteasome System ◽

Misfolded Proteins ◽

Critical Pathway ◽

Molecular Control ◽

Cytosolic Proteins ◽

Ubiquitination Machinery

The accumulation of misfolded proteins is associated with numerous degenerative conditions, cancers and genetic diseases. These pathological imbalances in protein homeostasis (termed proteostasis), result from the improper triage and disposal of damaged and defective proteins from the cell. The ubiquitin-proteasome system is a key pathway for the molecular control of misfolded cytosolic proteins, co-opting a cascade of ubiquitin ligases to direct terminally damaged proteins to the proteasome via modification with chains of the small protein, ubiquitin. Despite the evidence for ubiquitination in this critical pathway, the precise complement of ubiquitin ligases and deubiquitinases that modulate this process remains under investigation. Whilst chaperones act as the first line of defence against protein misfolding, the ubiquitination machinery has a pivotal role in targeting terminally defunct cytosolic proteins for destruction. Recent work points to a complex assemblage of chaperones, ubiquitination machinery and subcellular quarantine as components of the cellular arsenal against proteinopathies. In this review, we examine the contribution of these pathways and cellular compartments to the maintenance of the cytosolic proteome. Here we will particularly focus on the ubiquitin code and the critical enzymes which regulate misfolded proteins in the cytosol, the molecular point of origin for many neurodegenerative and genetic diseases.

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Eukaryotic Translation Initiation Factor 4E Activity Is Modulated by HOXA9 at Multiple Levels

Molecular and Cellular Biology ◽

10.1128/mcb.25.3.1100-1112.2005 ◽

2005 ◽

Vol 25 (3) ◽

pp. 1100-1112 ◽

Cited By ~ 59

Author(s):

Ivan Topisirovic ◽

Alex Kentsis ◽

Jacqueline M. Perez ◽

Monica L. Guzman ◽

Craig T. Jordan ◽

...

Keyword(s):

Translation Initiation ◽

Nuclear Export ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Homeodomain Protein ◽

Translation Efficiency ◽

Eukaryotic Translation Initiation Factor ◽

Eukaryotic Translation ◽

Eukaryotic Translation Initiation ◽

Multiple Levels

ABSTRACT The eukaryotic translation initiation factor 4E (eIF4E) alters gene expression on multiple levels. In the cytoplasm, eIF4E acts in the rate-limiting step of translation initiation. In the nucleus, eIF4E facilitates nuclear export of a subset of mRNAs. Both of these functions contribute to eIF4E's ability to oncogenically transform cells. We report here that the homeodomain protein, HOXA9, is a positive regulator of eIF4E. HOXA9 stimulates eIF4E-dependent export of cyclin D1 and ornithine decarboxylase (ODC) mRNAs in the nucleus, as well as increases the translation efficiency of ODC mRNA in the cytoplasm. These activities depend on direct interactions of HOXA9 with eIF4E and are independent of the role of HOXA9 in transcription. At the biochemical level, HOXA9 mediates these effects by competing with factors that repress eIF4E function, in particular the proline-rich homeodomain PRH/Hex. This competitive mechanism of eIF4E regulation is disrupted in a subset of leukemias, where HOXA9 displaces PRH from eIF4E, thereby contributing to eIF4E's dysregulation. In regard to these results and our previous finding that ∼200 homeodomain proteins contain eIF4E binding sites, we propose that homeodomain modulation of eIF4E activity is a novel means through which this family of proteins implements their effects on growth and development.

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Misfolded Proteins Recognition Strategies of E3 Ubiquitin Ligases and Neurodegenerative Diseases

Molecular Neurobiology ◽

10.1007/s12035-012-8351-0 ◽

2012 ◽

Vol 47 (1) ◽

pp. 302-312 ◽

Cited By ~ 23

Author(s):

Deepak Chhangani ◽

Nihar Ranjan Jana ◽

Amit Mishra

Keyword(s):

Neurodegenerative Diseases ◽

Ubiquitin Ligases ◽

Misfolded Proteins ◽

E3 Ubiquitin Ligases

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Particle characterization of CVD tungsten metallization for 64 MBIT DRAM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100088798 ◽

1991 ◽

Vol 49 ◽

pp. 896-897

Author(s):

Marylyn Bennett-Lilley ◽

Thomas T.H. Fu ◽

David D. Yin ◽

R. Allen Bowling

Keyword(s):

Chemical Vapor ◽

Device Performance ◽

Particle Characterization ◽

Particle Generation ◽

Tungsten Hexafluoride ◽

Homogeneous Particle ◽

Multiple Levels ◽

Circuit Density ◽

Sources Of Contamination

Chemical Vapor Deposition (CVD) tungsten metallization is used to increase VLSI device performance due to its low resistivity, and improved reliability over other metallization schemes. Because of its conformal nature as a blanket film, CVD-W has been adapted to multiple levels of metal which increases circuit density. It has been used to fabricate 16 MBIT DRAM technology in a manufacturing environment, and is the metallization for 64 MBIT DRAM technology currently under development. In this work, we investigate some sources of contamination. One possible source of contamination is impurities in the feed tungsten hexafluoride (WF6) gas. Another is particle generation from the various reactor components. Another generation source is homogeneous particle generation of particles from the WF6 gas itself. The purpose of this work is to investigate and analyze CVD-W process-generated particles, and establish a particle characterization methodology.

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