Protein quality control at the inner nuclear membrane

Khmelinskii, Anton; Blaszczak, Ewa; Pantazopoulou, Marina; Fischer, Bernd; Omnus, Deike J.; Dez, Gaëlle Le; Brossard, Audrey; Gunnarsson, Alexander; Barry, Joseph D.; Meurer, Matthias; Kirrmaier, Daniel; Boone, Charles; Huber, Wolfgang; Rabut, Gwénaël; Ljungdahl, Per O.; Knop, Michael

doi:10.1038/nature14096

Cited by 187 publications

(256 citation statements)

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“…In yeast, two E3 ubiquitin ligase complexes are known to localize to the INM and target nucleoplasmic and INM substrates: the Doa10 complex (6) and the Asi RING finger protein complex (56,57). No mammalian INM-embedded ubiquitin ligases have been identified to date.…”

Section: Discussionmentioning

confidence: 99%

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation

Zattas

Berk

Kreft

et al. 2016

Journal of Biological Chemistry

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Specific proteins are modified by ubiquitin at the endoplasmic reticulum (ER) and are degraded by the proteasome, a process referred to as ER-associated protein degradation. In Saccharomyces cerevisiae, two principal ER-associated protein degradation ubiquitin ligases (E3s) reside in the ER membrane, Doa10 and Hrd1. The membrane-embedded Doa10 functions in the degradation of substrates in the ER membrane, nuclear envelope, cytoplasm, and nucleoplasm. How most E3 ligases, including Doa10, recognize their protein substrates remains poorly understood. Here we describe a previously unappreciated but highly conserved C-terminal element (CTE) in Doa10; this cytosolically disposed 16-residue motif follows the final transmembrane helix. A conserved CTE asparagine residue is required for ubiquitylation and degradation of a subset of Doa10 substrates. Such selectivity suggests that the Doa10 CTE is involved in substrate discrimination and not general ligase function. Functional conservation of the CTE was investigated in the human ortholog of Doa10, MARCH6 (TEB4), by analyzing MARCH6 autoregulation of its own degradation. Mutation of the conserved Asn residue (N890A) in the MARCH6 CTE stabilized the normally short lived enzyme to the same degree as a catalytically inactivating mutation (C9A). We also report the localization of endogenous MARCH6 to the ER using epitope tagging of the genomic MARCH6 locus by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. These localization and CTE analyses support the inference that MARCH6 and Doa10 are functionally similar. Moreover, our results with the yeast enzyme suggest that the CTE is involved in the recognition and/or ubiquitylation of specific protein substrates.Selective protein degradation in eukaryotic cells is largely carried out by the ubiquitin-proteasome system. Proteins identified for degradation by the ubiquitin-proteasome system are covalently modified with ubiquitin, a highly conserved 76-residue protein (1, 2). In most cases, ubiquitin is conjugated to the target protein via an amide linkage between its C-terminal glycine and ⑀-amino groups on substrate lysine residues. Ubiquitylation requires an enzyme cascade beginning with an E1 ubiquitin-activating enzyme, which adenylates the ubiquitin C-terminal carboxyl group and subsequently forms a high energy thioester bond with it. Ubiquitin is then transferred from the E1 active site cysteine to a cysteine in an E2 ubiquitinconjugating enzyme. Finally, an E3 ubiquitin ligase mediates ubiquitin transfer from the E2 to the target protein. E3s come in several mechanistically distinct classes, but the most abundant are the RING E3s. The RING domain is characterized by a set of Cys and His residues that coordinate a pair of zinc ions; the RING directly contacts the E2-ubiquitin thioester-linked complex and promotes ubiquitin transfer to a substrate (3). Polyubiquitin chains are formed when the C terminus of one donor ubiquitin is conjugated to one of seven lysine residues, or the ...

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Section: Discussionmentioning

confidence: 99%

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation

Zattas

Berk

Kreft

et al. 2016

Journal of Biological Chemistry

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“…In addition to endogenous resident INM proteins, many membrane proteins that normally reside in other intracellular locations are apparently able to gain access to the INM by leaking past nuclear pore complexes (Foresti et al, 2014;Khmelinskii et al, 2014;Popken et al, 2015). Both resident and mislocalized INM proteins are subject to turnover (Khmelinskii et al, 2014;Omnus and Ljungdahl, 2014).…”

Section: Introductionmentioning

confidence: 99%

“…In addition to its well-established role in ERAD, Doa10 is able to pass the barrier of NPCs and function together with Ubc6 and Ubc7 in the INM to ubiquitylate multiple nuclear substrates, targeting them to nuclear proteasomes for degradation (Boban et al, 2014;Deng and Hochstrasser, 2006;Furth et al, 2011;Ravid et al, 2006). The second, and most recently characterized, INMAD pathway is defined by the Asi1-Asi3 E3 ubiquitin ligase complex (Khmelinskii et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

“…Both resident and mislocalized INM proteins are subject to turnover (Khmelinskii et al, 2014;Omnus and Ljungdahl, 2014). Currently, two quality control pathways are known to operate in association with the INM in yeast (Deng and Hochstrasser, 2006;Foresti et al, 2014;Khmelinskii et al, 2014). Analogous to the ERAD complexes, the INM-associated degradation (INMAD) complexes are defined by their core E3 ubiquitin ligases.…”

Section: Introductionmentioning

confidence: 99%

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Cdc48 and Ubx1 participate in a pathway associated with the inner nuclear membrane that governs Asi1 degradation

Pantazopoulou

Boban

Foisner

et al. 2016

Journal of Cell Science

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The nuclear envelope is a barrier comprising outer and inner membranes that separate the cytoplasm from the nucleoplasm. The two membranes have different physical characteristics and protein compositions. The processes governing the stability of inner nuclear membrane (INM) proteins are not well characterized. In Saccharomyces cerevisiae, the INM Asi1-Asi3 complex, principally composed of integral membrane proteins Asi1 and Asi3, is an E3 ubiquitin ligase. In addition to its well-documented function in endoplasmic reticulum (ER)-associated degradation, the Doa10 E3 ubiquitin ligase complex partially localizes to the INM. The Asi1-Asi3 and Doa10 complexes define independent INM-associated degradation (INMAD) pathways that target discrete sets of nuclear substrates for proteasomal degradation. Here, we report that Asi1 is rapidly turned over (t 1/2 ≤30 min). Its turnover depends on ubiquitinmediated degradation by nucleus-localized proteasomes, exhibiting a clear requirement for the E2 ubiquitin-conjugating enzyme Ubc7, Cue1 and the AAA ATPase Cdc48 and co-factor Ubx1. Asi1 turnover occurs largely independently of the Asi1-Asi3 or Doa10 complexes, indicating that it is subject to quality control at the INM in a manner distinct from that of the characterized INMAD pathways.

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“…Interestingly, recent work supports that an arm of ERAD exists at the INM. 33,34 The invaginations/intralumenal vesicles observed in strains containing vps4, npl4 and other alleles that impact NPC function, hints at another potential model where NPC assembly intermediates might be cleared through a vesicular intermediate in the NE lumen (Fig. 1D).…”

Section: Potential Models Of Aberrant Assembly Clearancementioning

confidence: 99%

ESCRTs breach the nuclear border

Webster

Lusk

2015

Nucleus

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T he endosomal sorting complexes required for transport (ESCRT) are best known for their role in sorting ubiquitylated membrane proteins into endosomes. The most ancient component of the ESCRT machinery is ESCRT-III, which is capable of oligomerizing into a helical filament that drives the invagination and scission of membranes aided by the AAA ATPase, Vps4, in several additional subcellular contexts. Our recent study broadens the work of ESCRT-III by identifying its role in a quality control pathway at the nuclear envelope (NE) that ensures the normal biogenesis of nuclear pore complexes (NPCs). Here, we will elaborate on how we envision this mechanism to progress and incorporate ESCRT-III into an emerging model of nuclear pore formation. Moreover, we speculate there are additional roles for the ESCRT-III machinery at the NE that broadly function to ensure its integrity and the maintenance of the nuclear compartment.

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Protein quality control at the inner nuclear membrane

Cited by 187 publications

References 42 publications

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation

Cdc48 and Ubx1 participate in a pathway associated with the inner nuclear membrane that governs Asi1 degradation

ESCRTs breach the nuclear border

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