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

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.

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Cycles of autoubiquitination and deubiquitination regulate the ERAD ubiquitin ligase Hrd1

eLife ◽

10.7554/elife.50903 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 8

Author(s):

Brian G Peterson ◽

Morgan L Glaser ◽

Tom A Rapoport ◽

Ryan D Baldridge

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Ubiquitin Ligase ◽

Ground State ◽

Inhibitory Effect ◽

The Other ◽

Misfolded Proteins ◽

Deubiquitinating Enzyme ◽

Transmembrane Segment

Misfolded proteins in the lumen of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol and polyubiquitinated before being degraded by the proteasome. The multi-spanning ubiquitin ligase Hrd1 forms the retrotranslocation channel and associates with three other membrane proteins (Hrd3, Usa1, Der1) of poorly defined function. The Hrd1 channel is gated by autoubiquitination, but how Hrd1 escapes degradation by the proteasome and returns to its inactive ground state is unknown. Here, we show that autoubiquitination of Hrd1 is counteracted by Ubp1, a deubiquitinating enzyme that requires its N-terminal transmembrane segment for activity towards Hrd1. The Hrd1 partner Hrd3 serves as a brake for autoubiquitination, while Usa1 attenuates Ubp1’s deubiquitination activity through an inhibitory effect of its UBL domain. These results lead to a model in which the Hrd1 channel is regulated by cycles of autoubiquitination and deubiquitination, reactions that are modulated by the other components of the Hrd1 complex.

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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.

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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.

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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.

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Ubiquitination in the ERAD Process

International Journal of Molecular Sciences ◽

10.3390/ijms21155369 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5369

Author(s):

Anna Lopata ◽

Andreas Kniss ◽

Frank Löhr ◽

Vladimir V. Rogov ◽

Volker Dötsch

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

The Other ◽

Misfolded Protein ◽

E3 Ligases ◽

Aaa Atpase ◽

A Cell ◽

Conjugating Enzymes ◽

The One ◽

Erad Pathway

In this review, we focus on the ubiquitination process within the endoplasmic reticulum associated protein degradation (ERAD) pathway. Approximately one third of all synthesized proteins in a cell are channeled into the endoplasmic reticulum (ER) lumen or are incorporated into the ER membrane. Since all newly synthesized proteins enter the ER in an unfolded manner, folding must occur within the ER lumen or co-translationally, rendering misfolding events a serious threat. To prevent the accumulation of misfolded protein in the ER, proteins that fail the quality control undergo retrotranslocation into the cytosol where they proceed with ubiquitination and degradation. The wide variety of misfolded targets requires on the one hand a promiscuity of the ubiquitination process and on the other hand a fast and highly processive mechanism. We present the various ERAD components involved in the ubiquitination process including the different E2 conjugating enzymes, E3 ligases, and E4 factors. The resulting K48-linked and K11-linked ubiquitin chains do not only represent a signal for degradation by the proteasome but are also recognized by the AAA+ ATPase Cdc48 and get in the process of retrotranslocation modified by enzymes bound to Cdc48. Lastly we discuss the conformations adopted in particular by K48-linked ubiquitin chains and their importance for degradation.

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PRP19: a novel spliceosomal component

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1876-1882.1993 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1876-1882

Author(s):

S C Cheng ◽

W Y Tarn ◽

T Y Tsao ◽

J Abelson

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Weight ◽

Amino Acid ◽

The Other ◽

Splicing Factor ◽

Growth Defect ◽

Splicing Factors ◽

Temperature Sensitive ◽

Amino Acid Residues ◽

Exact Function

We have isolated the gene of a splicing factor, PRP19, by complementation of the temperature-sensitive growth defect of the prp19 mutant of Saccharomyces cerevisiae. The gene encodes a protein of 502 amino acid residues of molecular weight 56,500, with no homology to sequences in the data base. Unlike other PRP proteins or mammalian splicing factors, the sequence of PRP19 has no discernible motif. Immunoprecipitation studies showed that PRP19 is associated with the spliceosome during the splicing reaction. Although the exact function of PRP19 remains unknown, PRP19 appears to be distinct from the other PRP proteins or other spliceosomal components.

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PRP19: a novel spliceosomal component.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1876 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1876-1882 ◽

Cited By ~ 41

Author(s):

S C Cheng ◽

W Y Tarn ◽

T Y Tsao ◽

J Abelson

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Weight ◽

Amino Acid ◽

The Other ◽

Splicing Factor ◽

Growth Defect ◽

Splicing Factors ◽

Temperature Sensitive ◽

Amino Acid Residues ◽

Exact Function

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Wss1 Is a SUMO-Dependent Isopeptidase That Interacts Genetically with the Slx5-Slx8 SUMO-Targeted Ubiquitin Ligase

Molecular and Cellular Biology ◽

10.1128/mcb.01649-09 ◽

2010 ◽

Vol 30 (15) ◽

pp. 3737-3748 ◽

Cited By ~ 38

Author(s):

Janet R. Mullen ◽

Chi-Fu Chen ◽

Steven J. Brill

Keyword(s):

Saccharomyces Cerevisiae ◽

Fusion Protein ◽

Ubiquitin Ligase ◽

Copy Number ◽

Dna Helicase ◽

Genome Integrity ◽

Growth Defect ◽

Sumo Fusion ◽

Isopeptidase Activity

ABSTRACT Protein sumoylation plays an important but poorly understood role in controlling genome integrity. In Saccharomyces cerevisiae, the Slx5-Slx8 SUMO-targeted Ub ligase appears to be needed to ubiquitinate sumoylated proteins that arise in the absence of the Sgs1 DNA helicase. WSS1, a high-copy-number suppressor of a mutant SUMO, was implicated in this pathway because it shares phenotypes with SLX5-SLX8 mutants, including a wss1Δ sgs1Δ synthetic-fitness defect. Here we show that Wss1, a putative metalloprotease, physically binds SUMO and displays in vitro isopeptidase activity on poly-SUMO chains. Like that of SLX5, overexpression of WSS1 suppresses sgs1Δ slx5Δ lethality and the ulp1ts growth defect. Interestingly, although Wss1 is relatively inactive on ubiquitinated substrates and poly-Ub chains, it efficiently deubiquitinates a Ub-SUMO isopeptide conjugate and a Ub-SUMO fusion protein. Wss1 was further implicated in Ub metabolism on the basis of its physical association with proteasomal subunits. The results suggest that Wss1 is a SUMO-dependent isopeptidase that acts on sumoylated substrates as they undergo proteasomal degradation.

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