The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases

Hwang, Cheol-Sang; Smerdel-Ramoya, Anna; Auerbach, Daniel; Varshavsky, Alexander

doi:10.1038/ncb2121

Cited by 134 publications

(161 citation statements)

References 71 publications

(174 reference statements)

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“…With deletion of the E2 ubiquitin-conjugating enzyme Rad6, the accumulation of a band of slightly slower migration was visible (Fig. 1D, lane 3), which indicated possible arginylation by Ate1 preceding ubiquitination by the Ubr1/Rad6 heterodimer and degradation (24,25). The m-b 2 -Mia40 core -C3S rescue in the Δrad6 and Δate1 cells indicated exposure of the m-form to the cytosolic quality control machinery supporting the release hypothesis.…”

Section: Significancementioning

confidence: 48%

Retro-translocation of mitochondrial intermembrane space proteins

Brągoszewski

Wasilewski

Sakowska

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

103

101

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The content of mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the intermembrane space by the mitochondrial intermembrane space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the mitochondrial proteome surveillance. Because most mitochondrial proteins originate in the cytosol, mitochondria had to develop a protein import system. Given the complex architecture of these organelles, with two membranes and two aqueous compartments, protein import and sorting require the cooperation of several pathways. The main entry gate for precursor proteins is the translocase of the outer mitochondrial membrane (TOM) complex. Upon entering mitochondria, proteins are routed to different sorting machineries (1-5).Reaching the final location is one step in the maturation of mitochondrial proteins that must be accompanied by their proper folding. The mitochondrial intermembrane space assembly (MIA) pathway for intermembrane space (IMS) proteins illustrates the importance of coupling these processes because this pathway links protein import with oxidative folding (6-10). Upon protein synthesis in the cytosol, the cysteine residues of IMS proteins remain in a reduced state, owing to the reducing properties of the cytosolic environment (11,12). After entering the TOM channel, precursor proteins are specifically recognized by Mia40 protein, and their cysteine residues are oxidized through the cooperative action of Mia40 and Erv1 proteins (7,(13)(14)(15)(16)(17). Mia40 is a receptor, folding catalyst, and disulfide carrier, and the Erv1 protein serves as a sulfhydryl oxidase. The oxidative folding is believed to provide a trapping mechanism that prevents the escape of proteins from the IMS back to the cytosol (10, 13, 18). Our initial result raised a possibility that the reverse process can also occur, as we observed the relocation of in vitro imported Tim8 from mitochondria to the incubation buffer (13). Thus, we sought to establish whether and how this process can proceed in the presence of the intact outer membrane (OM). Our study provides, to our knowledge, the first characterization of the mitochondrial protein retro-translocation. The protein retro-translocation serves as a regulatory and quality control mechanism for th...

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Section: Significancementioning

confidence: 48%

Retro-translocation of mitochondrial intermembrane space proteins

Brągoszewski

Wasilewski

Sakowska

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

103

101

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“…The Arg/N-end rule pathway targets specific unacetylated N-terminal residues (Fig. 1A) (17,25). The primary destabilizing N-terminal residues Arg, Lys, His, Leu, Phe, Tyr, Trp, and Ile are directly recognized by E3 N-recognins, whereas N-terminal Asp, Glu, Asn, Gln, and Cys function as destabilizing residues through their preliminary modifications.…”

mentioning

confidence: 99%

The N-end rule pathway counteracts cell death by destroying proapoptotic protein fragments

Piatkov

Brower

Varshavsky

2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

124

195

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In the course of apoptosis, activated caspases cleave ∼500 to ∼1,000 different proteins in a mammalian cell. The dynamics of apoptosis involve a number of previously identified, caspase-generated proapoptotic protein fragments, defined as those that increase the probability of apoptosis. In contrast to activated caspases, which can be counteracted by inhibitor of apoptosis proteins, there is little understanding of antiapoptotic responses to proapoptotic protein fragments. One possibility is the regulation of proapoptotic fragments through their selective degradation. The previously identified proapoptotic fragments Cys-RIPK1, Cys-TRAF1, Asp-BRCA1, Leu-LIMK1, Tyr-NEDD9, Arg-BID, Asp-BCL XL , Arg-BIM EL , Asp-EPHA4, and Tyr-MET bear destabilizing N-terminal residues. Tellingly, the destabilizing nature (but not necessarily the actual identity) of N-terminal residues of proapoptotic fragments was invariably conserved in evolution. Here, we show that these proapoptotic fragments are short-lived substrates of the Arg/N-end rule pathway. Metabolic stabilization of at least one such fragment, Cys-RIPK1, greatly augmented the activation of the apoptosis-inducing effector caspase-3. In agreement with this understanding, even a partial ablation of the Arg/N-end rule pathway in two specific N-end rule mutants is shown to sensitize cells to apoptosis. We also found that caspases can inactivate components of the Arg/N-end rule pathway, suggesting a mutual suppression between this pathway and proapoptotic signaling. Together, these results identify a mechanistically specific and functionally broad antiapoptotic role of the Arg/N-end rule pathway. In conjunction with other apoptosis-suppressing circuits, the Arg/N-end rule pathway contributes to thresholds that prevent a transient or otherwise weak proapoptotic signal from reaching the point of commitment to apoptosis.

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“…Ubr1-dependent ubiquitination of Fas2 requires ubiquitin-conjugating enzymes (E2). The major ubiquitin-conjugating enzyme known to cooperate with Ubr1 is Ubc2 (Rad6) (38). Pulse-chase analysis, seen in Fig.…”

Section: Degradation Of Orphan Fas2 Is Triggered By the Ubiquitin Ligmentioning

confidence: 99%

Quality Control of a Cytoplasmic Protein Complex

Scazzari¹,

Amm²,

Wolf

2015

Journal of Biological Chemistry

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Background: Superfluous subunits of protein complexes are degraded to avoid unwanted erroneous reactions. Results: A machinery consisting of Hsp70, Cdc48, and components of the ubiquitin-proteasome system eliminates orphan fatty acid synthase subunit Fas2. Conclusion: Chaperone motors and the ubiquitin-proteasome system balance the homeostasis of the fatty acid synthase complex. Significance: Selective proteolysis is a tool to regulate protein complex stoichiometry.

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The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases

Cited by 134 publications

References 71 publications

Retro-translocation of mitochondrial intermembrane space proteins

Retro-translocation of mitochondrial intermembrane space proteins

The N-end rule pathway counteracts cell death by destroying proapoptotic protein fragments

Quality Control of a Cytoplasmic Protein Complex

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