Rsp5/Nedd4 is the main ubiquitin ligase that targets cytosolic misfolded proteins following heat stress

Fang, Nancy N.; Chan, Gerard T.; Zhu, Mang; Comyn, Sophie A.; Persaud, Avinash K.; Deshaies, Raymond J.; Rotin, Daniela; Gsponer, Joerg; Mayor, Thibault

doi:10.1038/ncb3054

Cited by 169 publications

(193 citation statements)

References 66 publications

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“…3C). This is in contrast to the recent finding that Rsp5 is the main ubiquitin ligase that targets cytosolic proteins following heat stress (Fang et al, 2014). Thus, another ligase must be involved in forming polyubiquitin chains that direct Hem12 for proteasomal degradation.…”

Section: Figure 1 Hem12 Protein Is Monoubiuqitinated In Vivo and Thicontrasting

confidence: 53%

Hem12, an enzyme of heme biosynthesis pathway, is monoubiquitinated by Rsp5 ubiquitin ligase in yeast cells

et al. 2015

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Heme biosynthesis pathway is conserved in yeast and humans and hem12 yeast mutants mimic porphyria cutanea tarda (PCT), a hereditary human disease caused by mutations in the UROD gene. Even though mutations in other genes also affect UROD activity and predispose to sporadic PCT, the regulation of UROD is unknown. Here, we used yeast as a model to study regulation of Hem12 by ubiquitination and involvement of Rsp5 ubiquitin ligase in this process. We found that Hem12 is monoubiquitinated in vivo by Rsp5. Hem12 contains three conserved lysine residues located on the protein surface that can potentially be ubiquitinated and lysine K8 is close to the 36-LPEY-39 (PY) motif which binds WW domains of the Rsp5 ligase. The hem12-K8A mutation results in a defect in cell growth on a glycerol medium at 38oC but it does not affect the level of Hem12. The hem12-L36A,P37A mutations which destroy the PY motif result in a more profound growth defect on both, glycerol and glucose-containing media. However, after several passages on the glucose medium, the hem12-L36A,P37A cells adapt to the growth medium owing to higher expression of hem12-L36A,P37A gene and higher stability of the mutant Hem12-L36A,P37A protein. The Hem12 protein is downregulated upon heat stress in a Rsp5-independent way. Thus, Rsp5-dependent Hem12 monoubiquitination is important for its functioning, but not required for its degradation. Since Rsp5 has homologs among the Nedd4 family of ubiquitin ligases in humans, a similar regulation by ubiquitination might be also important for functioning of the human UROD.

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Section: Figure 1 Hem12 Protein Is Monoubiuqitinated In Vivo and Thicontrasting

confidence: 53%

Hem12, an enzyme of heme biosynthesis pathway, is monoubiquitinated by Rsp5 ubiquitin ligase in yeast cells

et al. 2015

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“…For example, the recruitment of ubiquitin-ligating factors with E4 activity playing a role in temperature response, such as Hul5 (44,45), could be postulated. Conversely, it has been shown that the 26S proteasome is mechanistically capable of degrading Lys-63-polyubiquitylated proteins in vitro (17).…”

Section: Discussionmentioning

confidence: 99%

Cold Temperature Induces the Reprogramming of Proteolytic Pathways in Yeast

Isasa

Suñer

Diaz

et al. 2016

Journal of Biological Chemistry

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Despite much evidence of the involvement of the proteasomeubiquitin signaling system in temperature stress response, the dynamics of the ubiquitylome during cold response has not yet been studied. Here, we have compared quantitative ubiquitylomes from a strain deficient in proteasome substrate recruitment and a reference strain during cold response. We have observed that a large group of proteins showing increased ubiquitylation in the proteasome mutant at low temperature is comprised by reverses suppressor of Ty-phenotype 5 (Rsp5)-regulated plasma membrane proteins. Analysis of internalization and degradation of plasma membrane proteins at low temperature showed that the proteasome becomes determinant for this process, whereas, at 30°C, the proteasome is dispensable. Moreover, our observations indicate that proteasomes have increased capacity to interact with lysine 63-polyubiquitylated proteins during low temperature in vivo. These unanticipated observations indicate that, during cold response, there is a proteolytic cellular reprogramming in which the proteasome acquires a role in the endocytic-vacuolar pathway.Proteasomal and endocytic-vacuolar pathways are responsible for the degradation of a large portion of proteins in eukaryotic cells, and their activities are fundamental for protein homeostasis. The endocytic-vacuolar pathway degrades internalized plasma membrane (PM) 3 proteins, proteins trafficking from Golgi to the endosome, and proteins from autophagic processes (1). The proteasome is the fate of cytosolic and nuclear proteins, co-translationally incomplete or misfolded proteins, partially aggregated proteins, and transmembrane proteins from the endoplasmic reticulum (2, 3). Despite the functional divergence between endocytic-vacuolar and proteasomal pathways, they share a key regulatory aspect: client proteins are modified by ubiquitin, and as a consequence ubiquitin-conjugating and ubiquitin-recognizing proteins act as receptors and regulators of both processes. The involvement of distinct ubiquitin-protein ligases, deubiquitylating factors, different polyubiquitin topologies, and specific recognition of ubiquitylated substrates appear to be key factors to ensure the independence of both pathways (1, 4, 5).In the initial steps of the endocytic pathway, yeast PM cargo proteins are mainly ubiquitylated by the Nedd4-type ubiquitin ligase Rsp5 (4, 6, 7). Ubiquitylated cargo proteins undergo endocytosis, a process orchestrated by epsins and epsin-like proteins, which possess two types of ubiquitin binding domains: ubiquitin-interacting motifs and ubiquitin-associated domains (8). Internalized proteins may exhibit monoubiquitylation or Lys-63-dependent polyubiquitylation (4, 7). In the endosome, ubiquitylated cargo is recruited to the ESCRT-0 complex constituted by Vps27 (Hrs) and Hse1 (STAM1/2). Both Vps27 and Hse1 proteins contain ubiquitin binding domains that mediate interaction with ubiquitylated cargo (9). During multivesicular body biogenesis, ubiquitin modification of cargo proteins is preserved...

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“…Data were filtered according to the following parameters: proteins with four or more spectral counts for the WT sample and greater than or equal to five-fold more counts in WT sample compared to TAP control (see Appendix Table S4 for complete unfiltered data). Identified proteins were analyzed for the presence of PY-like motifs [94]. were used in an in vitro ubiquitylation assay in the presence of RNF168.…”

Section: Discussionmentioning

confidence: 99%

“…Proteins shown passed the following filtering criteria in two biological replicates (Experiments 1 and 2): two or more spectral counts for the WT sample and greater than or equal to five-fold more counts in WT sample compared to TAP control. Identified proteins were analyzed for the presence of PY-like motifs [94]. …”

Section: In Vivo Validation Of Rsp5 Ubaitsmentioning

confidence: 99%

Ubiquitin‐Activated Interaction Traps ( UBAIT s) identify E3 ligase binding partners

et al. 2015

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We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (UbiquitinActivated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1-and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ-a histone protein involved in DNA repair-as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.

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Rsp5/Nedd4 is the main ubiquitin ligase that targets cytosolic misfolded proteins following heat stress

Cited by 169 publications

References 66 publications

Hem12, an enzyme of heme biosynthesis pathway, is monoubiquitinated by Rsp5 ubiquitin ligase in yeast cells

Hem12, an enzyme of heme biosynthesis pathway, is monoubiquitinated by Rsp5 ubiquitin ligase in yeast cells

Cold Temperature Induces the Reprogramming of Proteolytic Pathways in Yeast

Ubiquitin‐Activated Interaction Traps ( UBAIT s) identify E3 ligase binding partners

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