Disorder Targets Misorder in Nuclear Quality Control Degradation: A Disordered Ubiquitin Ligase Directly Recognizes Its Misfolded Substrates

San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in genome-wide association of TBP [that nucleates pre-initiation complex (PIC) formation for transcription initiation] and RNA polymerase II (Pol II). Our results reveal the roles of San1 in regulating TBP recruitment to the promoters and Pol II association with the coding sequences, and hence PIC formation and coordination of elongating Pol II, respectively. Consistently, transcription is altered in the absence of San1. Such transcriptional alteration is associated with impaired ubiquitylation and proteasomal degradation of Spt16 and gene association of Paf1, but not the incorporation of centromeric histone, Cse4, into the active genes in Δsan1 . Collectively, our results demonstrate distinct functions of a nuclear protein quality control factor in regulating the genome-wide PIC formation and elongating Pol II (and hence transcription), thus unraveling new gene regulatory mechanisms.

Download Full-text

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

Journal of Biological Chemistry ◽

10.1074/jbc.ac120.016191 ◽

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.

Download Full-text

Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase

Molecular Cell ◽

10.1016/j.molcel.2016.07.020 ◽

2016 ◽

Vol 63 (5) ◽

pp. 827-839 ◽

Cited By ~ 35

Author(s):

Annika Weber ◽

Itamar Cohen ◽

Oliver Popp ◽

Gunnar Dittmar ◽

Yuval Reiss ◽

...

Keyword(s):

Quality Control ◽

Ubiquitin Ligase ◽

Conjugating Enzymes

Download Full-text

In Vivo Action of the HRD Ubiquitin Ligase Complex: Mechanisms of Endoplasmic Reticulum Quality Control and Sterol Regulation

Molecular and Cellular Biology ◽

10.1128/mcb.21.13.4276-4291.2001 ◽

2001 ◽

Vol 21 (13) ◽

pp. 4276-4291 ◽

Cited By ~ 93

Author(s):

Richard G. Gardner ◽

Alexander G. Shearer ◽

Randolph Y. Hampton

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Ubiquitin Ligase ◽

Sequence Similarity ◽

High Specificity ◽

Specific Sequence ◽

Ubiquitin Ligase Complex ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzyme

ABSTRACT Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRDcomplex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent “misfolded” state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.

Download Full-text

The Cochaperone HspBP1 Inhibits the CHIP Ubiquitin Ligase and Stimulates the Maturation of the Cystic Fibrosis Transmembrane Conductance Regulator

Molecular Biology of the Cell ◽

10.1091/mbc.e04-04-0293 ◽

2004 ◽

Vol 15 (9) ◽

pp. 4003-4010 ◽

Cited By ~ 116

Author(s):

Simon Alberti ◽

Karsten Böhse ◽

Verena Arndt ◽

Anton Schmitz ◽

Jörg Höhfeld

Keyword(s):

Cystic Fibrosis ◽

Quality Control ◽

Ubiquitin Ligase ◽

Molecular Chaperones ◽

Protein Quality ◽

Regulatory Mechanism ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Ubiquitin Ligase Activity ◽

Cystic Fibrosis Transmembrane

The CHIP ubiquitin ligase turns molecular chaperones into protein degradation factors. CHIP associates with the chaperones Hsc70 and Hsp90 during the regulation of signaling pathways and during protein quality control, and directs chaperone-bound clients to the proteasome for degradation. Obviously, this destructive activity should be carefully controlled. Here, we identify the cochaperone HspBP1 as an inhibitor of CHIP. HspBP1 attenuates the ubiquitin ligase activity of CHIP when complexed with Hsc70. As a consequence, HspBP1 interferes with the CHIP-induced degradation of immature forms of the cystic fibrosis transmembrane conductance regulator (CFTR) and stimulates CFTR maturation. Our data reveal a novel regulatory mechanism that determines folding and degradation activities of molecular chaperones.

Download Full-text

Yos9p assists in the degradation of certain nonglycosylated proteins from the endoplasmic reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.e10-10-0832 ◽

2011 ◽

Vol 22 (16) ◽

pp. 2937-2945 ◽

Cited By ~ 24

Author(s):

Laura A. Jaenicke ◽

Holger Brendebach ◽

Matthias Selbach ◽

Christian Hirsch

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Ubiquitin Ligase ◽

Quantitative Proteomics ◽

Structural Defects ◽

Sterol Synthesis ◽

Mutant Variant ◽

Substrate Spectrum

The HRD ubiquitin ligase recognizes and ubiquitylates proteins of the endoplasmic reticulum that display structural defects. Here, we apply quantitative proteomics to characterize the substrate spectrum of the HRD complex. Among the identified substrates is Erg3p, a glycoprotein involved in sterol synthesis. We characterize Erg3p and demonstrate that the elimination of Erg3p requires Htm1p and Yos9p, two proteins that take part in the glycan-dependent turnover of aberrant proteins. We further show that the HRD ligase also mediates the breakdown of Erg3p and CPY* engineered to lack N-glycans. The degradation of these nonglycosylated substrates is enhanced by a mutant variant of Yos9p that has lost its affinity for oligosaccharides, indicating that Yos9p has a previously unrecognized role in the quality control of nonglycosylated proteins.

Download Full-text

Aberrant substrate engagement of the ER translocon triggers degradation by the Hrd1 ubiquitin ligase

The Journal of Cell Biology ◽

10.1083/jcb.201203061 ◽

2012 ◽

Vol 197 (6) ◽

pp. 761-773 ◽

Cited By ~ 43

Author(s):

Eric M. Rubenstein ◽

Stefan G. Kreft ◽

Wesley Greenblatt ◽

Robert Swanson ◽

Mark Hochstrasser

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Membrane Protein ◽

Ubiquitin Ligase ◽

Apolipoprotein B ◽

Secretory Pathway ◽

Protein A ◽

Proteasomal Degradation ◽

Membrane Localization ◽

General Role

Little is known about quality control of proteins that aberrantly or persistently engage the endoplasmic reticulum (ER)-localized translocon en route to membrane localization or the secretory pathway. Hrd1 and Doa10, the primary ubiquitin ligases that function in ER-associated degradation (ERAD) in yeast, target distinct subsets of misfolded or otherwise abnormal proteins based primarily on degradation signal (degron) location. We report the surprising observation that fusing Deg1, a cytoplasmic degron normally recognized by Doa10, to the Sec62 membrane protein rendered the protein a Hrd1 substrate. Hrd1-dependent degradation occurred when Deg1-Sec62 aberrantly engaged the Sec61 translocon channel and underwent topological rearrangement. Mutations that prevent translocon engagement caused a reversion to Doa10-dependent degradation. Similarly, a variant of apolipoprotein B, a protein known to be cotranslocationally targeted for proteasomal degradation, was also a Hrd1 substrate. Hrd1 therefore likely plays a general role in targeting proteins that persistently associate with and potentially obstruct the translocon.

Download Full-text

A Nucleus-based Quality Control Mechanism for Cytosolic Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e10-02-0111 ◽

2010 ◽

Vol 21 (13) ◽

pp. 2117-2127 ◽

Cited By ~ 110

Author(s):

Rupali Prasad ◽

Shinichi Kawaguchi ◽

Davis T.W. Ng

Keyword(s):

Quality Control ◽

Control Systems ◽

Ubiquitin Ligase ◽

Nuclear Import ◽

Control Mechanism ◽

E3 Ubiquitin Ligase ◽

26S Proteasome ◽

Cytosolic Proteins ◽

Hsp70 Chaperone ◽

Quality Control Mechanism

Intracellular quality control systems monitor protein conformational states. Irreversibly misfolded proteins are cleared through specialized degradation pathways. Their importance is underscored by numerous pathologies caused by aberrant proteins. In the cytosol, where most proteins are synthesized, quality control remains poorly understood. Stress-inducible chaperones and the 26S proteasome are known mediators but how their activities are linked is unclear. To better understand these mechanisms, a panel of model misfolded substrates was analyzed in detail. Surprisingly, their degradation occurs not in the cytosol but in the nucleus. Degradation is dependent on the E3 ubiquitin ligase San1p, known previously to direct the turnover of damaged nuclear proteins. A second E3 enzyme, Ubr1p, augments this activity but is insufficient by itself. San1p and Ubr1p are not required for nuclear import of substrates. Instead, the Hsp70 chaperone system is needed for efficient import and degradation. These data reveal a new function of the nucleus as a compartment central to the quality control of cytosolic proteins.

Download Full-text