scholarly journals CAT tails drive on- and off-ribosome degradation of stalled polypeptides

2018 ◽  
Author(s):  
Cole S. Sitron ◽  
Onn Brandman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Ubiquitin Ligase ◽  
Ribosomal Subunit ◽  
Protein Homeostasis ◽  
Large Ribosomal Subunit ◽  
Backup Route ◽  
Carboxy Terminal

SummaryStalled translation produces incomplete, ribosome-associated polypeptides that Ribosome-associated Quality Control (RQC) targets for degradation via the ubiquitin ligase Ltn1. During this process, the Rqc2 protein and large ribosomal subunit elongate stalled polypeptides with carboxy-terminal alanine and threonine residues (CAT tails). Failure to degrade CAT-tailed proteins disrupts global protein homeostasis, as CAT-tailed proteins aggregate and sequester chaperones. Why cells employ such a potentially toxic process during RQC is unclear. Here, we developed quantitative techniques to assess how CAT tails affect stalled polypeptide degradation in Saccharomyces cerevisiae. We found that CAT tails improve Ltn1’s efficiency in targeting structured polypeptides, which are otherwise poor Ltn1 substrates. If Ltn1 fails, CAT tails undergo a backup route of ubiquitylation off the ribosome, mediated by the ubiquitin ligase Hul5. Thus, CAT tails functionalize the carboxy-termini of stalled polypeptides to drive their degradation on and off the ribosome.

scholarly journals Aggregation of CAT tails blocks their degradation and causes proteotoxicity in S. cerevisiae

2019 ◽  
Author(s):  
Cole S. Sitron ◽  
Joseph H. Park ◽  
Jenna M. Giafaglione ◽  
Onn Brandman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Ribosomal Subunit ◽  
E3 Ligase ◽  
Protein Homeostasis ◽  
Large Ribosomal Subunit ◽  
Proteotoxic Stress ◽  
Chemical Tools ◽  
Carboxy Terminal

AbstractThe Ribosome-associated Quality Control (RQC) pathway co-translationally marks incomplete polypeptides from stalled translation with two signals that trigger their proteasome-mediated degradation. The E3 ligase Ltn1 adds ubiquitin and Rqc2 directs the large ribosomal subunit to append carboxy-terminal alanine and threonine residues (CAT tails). When excessive amounts of incomplete polypeptides evade Ltn1, CAT-tailed proteins accumulate and can self-associate into aggregates. CAT tail aggregation has been hypothesized to either protect cells by sequestering potentially toxic incomplete polypeptides or harm cells by disrupting protein homeostasis. To distinguish between these possibilities, we modulated CAT tail aggregation in Saccharomyces cerevisiae with genetic and chemical tools to analyze CAT tails in aggregated and un-aggregated states. We found that enhancing CAT tail aggregation induces proteotoxic stress and antagonizes degradation of CAT-tailed proteins, while inhibiting aggregation reverses these effects. Our findings suggest that CAT tail aggregation harms RQC-compromised cells and that preventing aggregation can mitigate this toxicity.

scholarly journals Overlapping function of Hrd1 and Ste24 in translocon quality control provides robust channel surveillance

2020 ◽  
Vol 295 (47) ◽  
pp. 16113-16120
Author(s):  
Avery M. Runnebohm ◽  
Kyle A. Richards ◽  
Courtney Broshar Irelan ◽  
Samantha M. Turk ◽  
Halie E. Vitali ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Ubiquitin Ligase ◽  
Biological Membranes ◽  
The Other ◽  
Growth Defect ◽  
Zinc Metalloprotease

Translocation of proteins across biological membranes is essential for life. Proteins that clog the endoplasmic reticulum (ER) translocon prevent the movement of other proteins into the ER. Eukaryotes have multiple translocon quality control (TQC) mechanisms to detect and destroy proteins that persistently engage the translocon. TQC mechanisms have been defined using a limited panel of substrates that aberrantly occupy the channel. The extent of substrate overlap among TQC pathways is unknown. In this study, we found that two TQC enzymes, the ER-associated degradation ubiquitin ligase Hrd1 and zinc metalloprotease Ste24, promote degradation of characterized translocon-associated substrates of the other enzyme in Saccharomyces cerevisiae. Although both enzymes contribute to substrate turnover, our results suggest a prominent role for Hrd1 in TQC. Yeast lacking both Hrd1 and Ste24 exhibit a profound growth defect, consistent with overlapping function. Remarkably, two mutations that mildly perturb post-translational translocation and reduce the extent of aberrant translocon engagement by a model substrate diminish cellular dependence on TQC enzymes. Our data reveal previously unappreciated mechanistic complexity in TQC substrate detection and suggest that a robust translocon surveillance infrastructure maintains functional and efficient translocation machinery.

scholarly journals Innate immunity to yeast prions: Btn2p and Cur1p curing of the [URE3] prion is prevented by 60S ribosomal protein deficiency or ubiquitin/proteasome system overactivity

Genetics ◽  
2021 ◽  
Author(s):  
Evgeny E Bezsonov ◽  
Herman K Edskes ◽  
Reed B Wickner
Keyword(s):  
Ubiquitin Ligase ◽  
Ribosomal Subunit ◽  
Ubiquitin Proteasome System ◽  
Negative Regulator ◽  
Large Ribosomal Subunit ◽  
Seed Number ◽  
Wild Type ◽  
Yeast Prions ◽  
In The Wild ◽  
Ubiquitin Proteasome

Abstract [URE3] is an amyloid-based prion of Ure2p, a negative regulator of poor nitrogen source catabolism in Saccharomyces cerevisiae. Overproduced Btn2p or its paralog Cur1p, in processes requiring Hsp42, cure the [URE3] prion. Btn2p cures by collecting Ure2p amyloid filaments at one place in the cell. We find that rpl4aΔ, rpl21aΔ, rpl21bΔ, rpl11bΔ and rpl16bΔ (large ribosomal subunit proteins) or ubr2Δ (ubiquitin ligase targeting Rpn4p, an activator of proteasome genes) reduce curing by overproduced Btn2p or Cur1p. Impaired curing in ubr2Δ or rpl21bΔ is restored by an rpn4Δ mutation. No effect of rps14aΔ or rps30bΔ on curing was observed, indicating that 60S subunit deficiency specifically impairs curing. Levels of Hsp42p, Sis1p or Btn3p are unchanged in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Overproduction of Cur1p or Btn2p was enhanced in rpn4Δ and hsp42Δ mutants, lower in ubr2Δ strains, and restored to above wild type levels in rpn4Δ ubr2Δ strains. As in the wild-type, Ure2N-GFP colocalizes with Btn2-RFP in rpl4aΔ, rpl21bΔ or ubr2Δ strains, but not in hsp42Δ. Btn2p/Cur1p overproduction cures [URE3] variants with low seed number, but seed number is not increased in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Knockouts of genes required for the protein sorting function of Btn2p did not affect curing of [URE3], nor did inactivation of the Hsp104 prion-curing activity. Overactivity of the ubiquitin/proteasome system, resulting from 60S subunit deficiency or ubr2Δ, may impair Cur1p and Btn2p curing of [URE3] by degrading Cur1p, Btn2p or another component of these curing systems.

scholarly journals Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

2014 ◽  
Vol 111 (45) ◽  
pp. 15981-15986 ◽  
Author(s):  
Dmitry Lyumkis ◽  
Dario Oliveira dos Passos ◽  
Erich B. Tahara ◽  
Kristofor Webb ◽  
Eric J. Bennett ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Ribosomal Subunit ◽  
Protein Quality Control ◽  
Structural Basis ◽  
Large Ribosomal Subunit ◽  
Control Complex

scholarly journals Loss of protein quality control gene UBR1 sensitizes Saccharomyces cerevisiae to the aminoglycoside hygromycin B

Fine Focus ◽  
2020 ◽  
Vol 6 (1) ◽  
pp. 76-83
Author(s):  
Avery M. Runnebohm ◽  
Melissa D. Evans ◽  
Adam E. Richardson ◽  
Samantha M. Turk ◽  
James B. Olesen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Ubiquitin Ligase ◽  
Protein Quality ◽  
Protein Quality Control ◽  
Eukaryotic Cells ◽  
Hygromycin B ◽  
Eukaryotic Microorganism ◽  
Human Enzyme ◽  
A Site

Ubr1 is a conserved ubiquitin ligase involved in the degradation of aberrant proteins in eukaryotic cells. The human enzyme is found mutated in patients with Johanson-Blizzard syndrome. We hypothesized that Ubr1 is necessary for optimal cellular fitness in conditions associated with elevated abundance of aberrant and misfolded proteins. Indeed, we found that loss of Ubr1 in the model eukaryotic microorganism Saccharomyces cerevisiae strongly sensitizes cells to hygromycin B, which reduces translational fidelity by causing ribosome A site distortion. Our results are consistent with a prominent role for Ubr1 in protein quality control. We speculate that disease manifestations in patients with Johanson-Blizzard syndrome are linked, at least in part, to defects in protein quality control caused by loss of Ubr1 function.

Acidic proteins of the large ribosomal subunit in Saccharomyces cerevisiae. Effect of phosphorylation

Biochemistry ◽  
1984 ◽  
Vol 23 (2) ◽  
pp. 390-396 ◽  
Author(s):  
F. Juan Vidales ◽  
M. T. Saenz Robles ◽  
J. P. G. Ballesta
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Subunit ◽  
Large Ribosomal Subunit ◽  
Acidic Proteins

scholarly journals A conserved quality-control pathway that mediates degradation of unassembled ribosomal proteins

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Min-Kyung Sung ◽  
Tanya R Porras-Yakushi ◽  
Justin M Reitsma ◽  
Ferdinand M Huber ◽  
Michael J Sweredoski ◽  
...  
Keyword(s):  
Quality Control ◽  
Ribosomal Protein ◽  
Ubiquitin Ligase ◽  
Ribosomal Proteins ◽  
Protein Quality ◽  
Ubiquitin Proteasome System ◽  
Protein Homeostasis ◽  
System Quality ◽  
Ubiquitin Proteasome ◽  
E2 Enzymes

Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes.

scholarly journals Quality Control of Plasma Membrane Proteins by Saccharomyces cerevisiae Nedd4-Like Ubiquitin Ligase Rsp5p under Environmental Stress Conditions

2014 ◽  
Vol 13 (9) ◽  
pp. 1191-1199 ◽  
Author(s):  
Takeki Shiga ◽  
Nobuyuki Yoshida ◽  
Yuko Shimizu ◽  
Etsuko Suzuki ◽  
Toshiya Sasaki ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Ubiquitin Ligase ◽  
Intracellular Trafficking ◽  
Environmental Stresses ◽  
Stress Conditions ◽  
Plasma Membrane Proteins ◽  
Content Type

ABSTRACTInSaccharomyces cerevisiae, when a rich nitrogen source such as ammonium is added to the culture medium, the general amino acid permease Gap1p is ubiquitinated by the yeast Nedd4-like ubiquitin ligase Rsp5p, followed by its endocytosis to the vacuole. The arrestin-like Bul1/2p adaptors for Rsp5p specifically mediate this process. In this study, to investigate the downregulation of Gap1p in response to environmental stresses, we determined the intracellular trafficking of Gap1p under various stress conditions. An increase in the extracellular ethanol concentration induced ubiquitination and trafficking of Gap1p from the plasma membrane to the vacuole in wild-type cells, whereas Gap1p remained stable on the plasma membrane under the same conditions inrsp5A401Eand Δend3cells. A14C-labeled citrulline uptake assay using a nonubiquitinated form of Gap1p (Gap1pK9R/K16R) revealed that ethanol stress caused a dramatic decrease of Gap1p activity. These results suggest that Gap1p is inactivated and ubiquitinated by Rsp5p for endocytosis whenS. cerevisiaecells are exposed to a high concentration of ethanol. It is noteworthy that this endocytosis occurs in a Bul1/2p-independent manner, whereas ammonium-triggered downregulation of Gap1p was almost completely inhibited in Δbul1/2cells. We also found that other environmental stresses, such as high temperature, H2O2, and LiCl, also promoted endocytosis of Gap1p. Similar intracellular trafficking caused by ethanol occurred in other plasma membrane proteins (Agp1p, Tat2p, and Gnp1p). Our findings suggest that stress-induced quality control is a common process requiring Rsp5p for plasma membrane proteins in yeast.

High levels of the mitochondrial large ribosomal subunit protein 40 prevent loss of mitochondrial DNA in nullmmf1 Saccharomyces cerevisiae cells

Yeast ◽  
2004 ◽  
Vol 21 (7) ◽  
pp. 539-548 ◽  
Author(s):  
Rosita Accardi ◽  
Ellinor Oxelmark ◽  
Nicolas Jauniaux ◽  
Vito de Pinto ◽  
Antonio Marchini ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Ribosomal Subunit ◽  
Large Ribosomal Subunit ◽  
Subunit Protein ◽  
Ribosomal Subunit Protein
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