scholarly journals EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired

2020 ◽  
Vol 21 (10) ◽  
pp. 3468 ◽  
Author(s):  
Marioara Chiritoiu ◽  
Gabriela N. Chiritoiu ◽  
Cristian V. A. Munteanu ◽  
Florin Pastrama ◽  
N. Erwin Ivessa ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Endoplasmic Reticulum ◽  
Protein Degradation ◽  
Protein Interaction ◽  
Proteasomal Degradation ◽  
Misfolded Proteins ◽  
Misfolded Protein ◽  
Protein Protein Interaction ◽  
Pathological Conditions ◽  
Er Homeostasis

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the main mechanism of targeting ER proteins for degradation to maintain homeostasis, and perturbations of ERAD lead to pathological conditions. ER-degradation enhancing α-mannosidase-like (EDEM1) was proposed to extract terminally misfolded proteins from the calnexin folding cycle and target them for degradation by ERAD. Here, using mass-spectrometry and biochemical methods, we show that EDEM1 is found in auto-regulatory complexes with ERAD components. Moreover, the N-terminal disordered region of EDEM1 mediates protein–protein interaction with misfolded proteins, whilst the absence of this domain significantly impairs their degradation. We also determined that overexpression of EDEM1 can induce degradation, even when proteasomal activity is severely impaired, by promoting the formation of aggregates, which can be further degraded by autophagy. Therefore, we propose that EDEM1 maintains ER homeostasis and mediates ERAD client degradation via autophagy when either dislocation or proteasomal degradation are impaired.

scholarly journals Ubiquitin conjugation triggers misfolded protein sequestration into quality control foci when Hsp70 chaperone levels are limiting

2013 ◽  
Vol 24 (13) ◽  
pp. 2076-2087 ◽  
Author(s):  
Ayala Shiber ◽  
William Breuer ◽  
Michael Brandeis ◽  
Tommer Ravid
Keyword(s):  
Quality Control ◽  
Heat Shock Protein 70 ◽  
Inclusion Bodies ◽  
Amyloid Plaques ◽  
Model System ◽  
Misfolded Proteins ◽  
Misfolded Protein ◽  
Pathological Conditions ◽  
Hsp70 Chaperone ◽  
Ubiquitin Conjugation

Ubiquitin accumulation in amyloid plaques is a pathological marker observed in the vast majority of neurodegenerative diseases, yet ubiquitin function in these inclusions is controversial. It has been suggested that ubiquitylated proteins are directed to inclusion bodies under stress conditions, when both chaperone-mediated refolding and proteasomal degradation are compromised or overwhelmed. Alternatively, ubiquitin and chaperones may be recruited to preformed inclusions to promote their elimination. We address this issue using a yeast model system, based on expression of several mildly misfolded degradation substrates in cells with altered chaperone content. We find that the heat shock protein 70 (Hsp70) chaperone pair Ssa1/Ssa2 and the Hsp40 cochaperone Sis1 are essential for degradation. Substrate ubiquitylation is strictly dependent on Sis1, whereas Ssa1 and Ssa2 are dispensable. Remarkably, in Ssa1/Ssa2-depleted cells, ubiquitylated substrates are sequestered into detergent-insoluble, Hsp42-positive inclusion bodies. Unexpectedly, sequestration is abolished by preventing substrate ubiquitylation. We conclude that Hsp40 is required for the targeting of misfolded proteins to the ubiquitylation machinery, whereas the decision to degrade or sequester ubiquitylated proteins is mediated by the Hsp70s. Accordingly, diminished Hsp70 levels, as observed in aging or certain pathological conditions, might be sufficient to trigger ubiquitin-dependent sequestration of partially misfolded proteins into inclusion bodies.

scholarly journals Split-TurboID enables contact-dependent proximity labeling in cells

2020 ◽  
Author(s):  
Kelvin F. Cho ◽  
Tess C. Branon ◽  
Sanjana Rajeev ◽  
Tanya Svinkina ◽  
Namrata D. Udeshi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interaction ◽  
Proteomic Analysis ◽  
Protein Protein Interaction ◽  
Conventional Fractionation ◽  
Contact Sites ◽  
Biochemical Fractionation ◽  
Versatile Tool ◽  
Endogenous Proteins ◽  
In Cells

AbstractProximity labeling (PL) catalyzed by promiscuous enzymes such as TurboID have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum (ER)-mitochondria contact sites, reconstituted TurboID catalyzed spatially-restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to ER-mitochondria contacts. We validated eight novel candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially-specific proximity labeling in cells.

scholarly journals ESCRT machinery mediates selective microautophagy of endoplasmic reticulum

2019 ◽  
Author(s):  
Jasmin A. Schäfer ◽  
Julia P. Schessner ◽  
Peter W. Bircham ◽  
Takuma Tsuji ◽  
Charlotta Funaya ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Lysosomal Membrane ◽  
Misfolded Proteins ◽  
Selective Autophagy ◽  
Escrt Machinery ◽  
Parallel Pathways ◽  
Mechanistic Understanding ◽  
Er Homeostasis ◽  
Er Whorls

ABSTRACTER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and ESCRT proteins as key components for micro-ER-phagy. Third, we demonstrate that macro- and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that ESCRT proteins directly function in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis.

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