Browse
Submit Data
Databases
API
Help

Dataset Information

10 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The Hrd1p ligase complex forms a linchpin between ER-lumenal substrate selection and Cdc48p recruitment.

ABSTRACT: Misfolded proteins of the endoplasmic reticulum (ER) are targeted to the cytoplasm for proteasomal degradation. Key components of this process are ER membrane-bound ubiquitin ligases. These ligases associate with the cytoplasmic AAA-ATPase Cdc48p/p97, which is thought to support the release of malfolded proteins from the ER. Here, we characterize a yeast protein complex containing the ubiquitin ligase Hrd1p and the ER membrane proteins Hrd3p and Der1p. Hrd3p binds malfolded proteins in the ER lumen enabling their delivery to downstream components. Therefore, we propose that Hrd3p acts as a substrate recruitment factor for the Hrd1p ligase complex. Hrd3p function is also required for the association of Cdc48p with Hrd1p. Moreover, our data demonstrate that recruitment of Cdc48p depends on substrate processing by the Hrd1p ligase complex. Thus, the Hrd1p ligase complex unites substrate selection in the ER lumen and polyubiquitination in the cytoplasm and links these processes to the release of ER proteins via the Cdc48p complex.

SUBMITTER: Gauss R

PROVIDER: S-EPMC1456945 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p.

Project description:Misfolded, luminal endoplasmic reticulum (ER) proteins are retrotranslocated into the cytosol and degraded by the ubiquitin/proteasome system. This ERAD-L pathway requires a protein complex consisting of the ubiquitin ligase Hrd1p, which spans the ER membrane multiple times, and the membrane proteins Hrd3p, Usa1p, and Der1p. Here, we show that Hrd1p is the central membrane component in ERAD-L; its overexpression bypasses the need for the other components of the Hrd1p complex. Hrd1p function requires its oligomerization, which in wild-type cells is facilitated by Usa1p. Site-specific photocrosslinking indicates that, at early stages of retrotranslocation, Hrd1p interacts with a substrate segment close to the degradation signal. This interaction follows the delivery of substrate through other ERAD components, requires the presence of transmembrane segments of Hrd1p, and depends on both the ubiquitin ligase activity of Hrd1p and the function of the Cdc48p ATPase complex. Our results suggest a model for how Hrd1p promotes polypeptide movement through the ER membrane.

| S-EPMC3026631 | biostudies-literature

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation.

Project description:The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.

| S-EPMC3315801 | biostudies-other

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

Project description: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.

| S-EPMC3373407 | biostudies-literature

Misfolded membrane proteins are specifically recognized by the transmembrane domain of the Hrd1p ubiquitin ligase.

Project description:Quality control pathways such as ER-associated degradation (ERAD) employ a small number of factors to specifically recognize a wide variety of protein substrates. Delineating the mechanisms of substrate selection is a principle goal in studying quality control. The Hrd1p ubiquitin ligase mediates ERAD of numerous misfolded proteins including soluble, lumenal ERAD-L and membrane-anchored ERAD-M substrates. We tested if the Hrd1p multispanning membrane domain was involved in ERAD-M specificity. In this work, we have identified site-directed membrane domain mutants of Hrd1p impaired only for ERAD-M and normal for ERAD-L. Furthermore, other Hrd1p variants were specifically deficient for degradation of individual ERAD-M substrates. Thus, the Hrd1p transmembrane region bears determinants of high specificity in the ERAD-M pathway. From in vitro and interaction studies, we suggest a model in which the Hrd1p membrane domain employs intramembrane residues to evaluate substrate misfolding, leading to selective ubiquitination of appropriate ERAD-M clients.

| S-EPMC2710143 | biostudies-literature

Erv26p-dependent export of alkaline phosphatase from the ER requires lumenal domain recognition.

Project description:Active sorting at the endoplasmic reticulum (ER) drives efficient export of fully folded secretory proteins into coat protein complex II (COPII) vesicles, whereas ER-resident and misfolded proteins are retained and/or degraded. A number of secretory proteins depend upon polytopic cargo receptors for linkage to the COPII coat and ER export. However, the mechanism by which cargo receptors recognize transport-competent cargo is poorly understood. Here we examine the sorting determinants required for export of yeast alkaline phosphatase (ALP) by its cargo receptor Erv26p. Analyses of ALP chimeras and mutants indicated that Erv26p recognizes sorting information in the lumenal domain of ALP. This lumenal domain sorting signal must be positioned near the inner leaflet of the ER membrane for Erv26p-dependent export. Moreover, only assembled ALP dimers were efficiently recognized by Erv26p while an ALP mutant blocked in dimer assembly failed to exit the ER and was subjected to ER-associated degradation. These results further refine sorting information for ER export of ALP and show that recognition of folded cargo by export receptors contributes to strict ER quality control.

| S-EPMC2957810 | biostudies-literature

Usa1p is required for optimal function and regulation of the Hrd1p endoplasmic reticulum-associated degradation ubiquitin ligase.

Project description:Usa1p is a recently discovered member of the HRD ubiquitin ligase complex. The HRD pathway is a conserved route of ubiquitin-dependent, endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous lumenal (ERAD-L) and membrane-anchored (ERAD-M) substrates. We have investigated Usa1p to understand its importance in HRD complex action. Usa1p was required for the optimal function of the Hrd1p E3 ubiquitin ligase; its loss caused deficient degradation of both membrane-associated and lumenal proteins. Furthermore, Usa1p functioned in regulation of Hrd1p by two mechanisms. First, Hrd1p self-degradation, which serves to limit the levels of uncomplexed E3, is absolutely dependent on Usa1p and the ubiquitin-like (Ubl) domain of Usa1p. We found that Usa1p allows Hrd1p degradation by promoting trans interactions between Hrd1p molecules. The Ubl domain of Usa1p was required specifically for Hrd1p self-ubiquitination but not for degradation of either ERAD-L or ERAD-M substrates. In addition, Usa1p was able to attenuate the activity-dependent toxicity of Hrd1p without compromising substrate degradation, indicating a separate role in ligase regulation that operates in parallel to stability control. Many of the described actions of Usa1p are distinct from those of Der1p, which is recruited to the HRD complex by Usa1p. Thus, this novel, conserved factor is broadly involved in the function and regulation of the HRD pathway of ERAD.

| S-EPMC2820741 | biostudies-literature

Pivotal role of the C2 domain of the Smurf1 ubiquitin ligase in substrate selection.

Project description:The C2-WW-HECT-type ubiquitin ligases Smurf1 and Smurf2 play a critical role in embryogenesis and adult bone homeostasis via regulation of bone morphogenetic protein, Wnt, and RhoA signaling pathways. The intramolecular interaction between C2 and HECT domains autoinhibits the ligase activity of Smurf2. However, the role of the Smurf1 C2 domain remains elusive. Here, we show that the C2-HECT autoinhibition mechanism is not observed in Smurf1, and instead its C2 domain functions in substrate selection. The Smurf1 C2 domain exerts a key role in localization to the plasma membrane and endows Smurf1 with differential activity toward RhoA versus Smad5 and Runx2. Crystal structure analysis reveals that the Smurf1 C2 domain possesses a typical anti-parallel β-sandwich fold. Examination of the sulfate-binding site analysis reveals two key lysine residues, Lys-28 and Lys-85, within the C2 domain that are important for Smurf1 localization at the plasma membrane, regulation on cell migration, and robust ligase activity toward RhoA, which further supports a Ca(2+)-independent localization mechanism for Smurf1. These findings demonstrate a previously unidentified role of the Smurf1 C2 domain in substrate selection and cellular localization.

| S-EPMC3089529 | biostudies-literature

ERAD and protein import defects in a sec61 mutant lacking ER-lumenal loop 7.

Project description:The Sec61 channel mediates protein translocation across the endoplasmic reticulum (ER) membrane during secretory protein biogenesis, and likely also during export of misfolded proteins for ER-associated degradation (ERAD). The mechanisms of channel opening for the different modes of translocation are not understood so far, but the position of the large ER-lumenal loop 7 of Sec61p suggests a decisive role.We show here that the Y345H mutation in L7 which causes diabetes in the mouse displays no ER import defects in yeast, but a delay in misfolded protein export. A complete deletion of L7 in Sec61p resulted in viable, cold- and tunicamycin-hypersensitive yeast cells with strong defects in posttranslational protein import of soluble proteins into the ER, and in ERAD of soluble substrates. Membrane protein ERAD was only moderately slower in sec61?L7 than in wildtype cells. Although Sec61?L7 channels were unstable in detergent, co-translational protein integration into the ER membrane, proteasome binding to Sec61?L7 channels, and formation of hetero-heptameric Sec complexes were not affected.We conclude that L7 of Sec61p is required for initiation of posttranslational soluble protein import into and misfolded soluble protein export from the ER, suggesting a key role for L7 in transverse gating of the Sec61 channel.

| S-EPMC3897919 | biostudies-literature

PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER.

Project description:Glycosylphosphatidylinositol (GPI) acts as a membrane anchor of many cell surface proteins. Its structure and biosynthetic pathway are generally conserved among eukaryotic organisms, with a number of differences. In particular, mammalian and protozoan mannosyltransferases needed for addition of the first mannose (GPI-MT-I) have different substrate specificities and are targets of species- specific inhibitors of GPI biosynthesis. GPI-MT-I, however, has not been molecularly characterized. Characterization of GPI-MT-I would also help to clarify the topology of GPI biosynthesis. Here, we report a human cell line defective in GPI-MT-I and the gene responsible, PIG-M. PIG-M encodes a new type of mannosyltransferase of 423 amino acids, bearing multiple transmembrane domains. PIG-M has a functionally important DXD motif, a characteristic of many glycosyltransferases, within a domain facing the lumen of the endoplasmic reticulum (ER), indicating that transfer of the first mannose to GPI occurs on the lumenal side of the ER membrane.

| S-EPMC140182 | biostudies-literature

The ER-associated degradation component Der1p and its homolog Dfm1p are contained in complexes with distinct cofactors of the ATPase Cdc48p.

Project description:Misfolded proteins in the endoplasmic reticulum (ER) are often degraded in the cytosol by a process called ER-associated protein degradation (ERAD). During ERAD in S. cerevisiae, the ATPase Cdc48p associates with Der1p, a putative component of a retro-translocation channel. Cdc48p also binds a homolog of Der1p, Dfm1p, that has no known function in ERAD. Here, we show that Der1p and Dfm1p are contained in distinct complexes. While the complexes share several ERAD components, only the Dfm1p complex contains the Cdc48p cofactors Ubx1p and Ubx7p, while the Der1p complex is enriched in Ufd1p. These data suggest distinct functions for the Der1p and Dfm1p complexes.

| S-EPMC2438607 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data