Endoplasmic reticulum (ER)-associated protein degradation by the ubiquitin-proteasome system requ... more Endoplasmic reticulum (ER)-associated protein degradation by the ubiquitin-proteasome system requires the dislocation of substrates from the ER into the cytosol. It has been speculated that a functional ubiquitin proteasome pathway is not only essential for proteolysis, but also for the preceding export step. Here, we show that short ubiquitin chains synthesized on proteolytic substrates are not sufficient to complete dislocation; the size of the chain seems to be a critical determinant. Moreover, our results suggest that the AAA proteins of the 26S proteasome are not directly involved in substrate export. Instead, a related AAA complex Cdc48, is required for ER-associated protein degradation upstream of the proteasome.
Mistakes are part of our world and constantly occurring. Due to transcriptional and translational... more Mistakes are part of our world and constantly occurring. Due to transcriptional and translational failures, genomic mutations or diverse stress conditions like oxidation or heat misfolded proteins are permanently produced in every compartment of the cell. As misfolded proteins in general lose their native function and tend to aggregate several cellular mechanisms have been evolved dealing with such potentially toxic protein species. Misfolded proteins are mostly recognized by chaperones on the basis of their exposed hydrophobic patches and, if unable to refold them to their native state, are targeted to proteolytic pathways. Most prominent are the ubiquitin-proteasome system and the autophagic vacuolar (lysosomal) system, eliminating misfolded proteins from the cellular environment. A major task of this quality control system is the specific recognition and separation of the misfolded from the correctly folded protein species and the folding intermediates, respectively, which are on the way to the correct folded state but exhibit properties of misfolded proteins. In this review we focus on the recognition process and subsequent degradation of misfolded proteins via the ubiquitin-proteasome system in the different cell compartments of eukaryotic cells. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.
Proteins enter the secretory pathway through the endoplasmic reticulum, which delivers properly f... more Proteins enter the secretory pathway through the endoplasmic reticulum, which delivers properly folded proteins to their site of action and contains a quality-control system to monitor and prevent abnormal proteins from being delivered. Many of these proteins are degraded by the cytoplasmic proteasome, which requires their retrograde transport to the cytoplasm. Based on a co-immunoprecipitation of major histocompatibility complex (MHC) class I heavy-chain breakdown intermediates with the translocon subunit Sec61p, it was speculated that Sec61p maybe involved in retrograde transport. Here we present functional evidence from genetic studies that Sec61p mediates retrograde transport of a mutated lumenal yeast carboxypeptidase ycsY (CPY*) in vivo. The endoplasmic reticulum lumenal chaperone BiP (Kar2p) and Sec63p, which are also subunits of the import machinery, are involved in export of CPY* to the cytosol. Thus our results demonstrate that retrograde transport of proteins is mediated ...
Proteins enter the secretory pathway through the endoplasmic reticulum, which delivers properly f... more Proteins enter the secretory pathway through the endoplasmic reticulum, which delivers properly folded proteins to their site of action and contains a quality-control system to monitor and prevent abnormal proteins from being delivered. Many of these proteins are degraded by the cytoplasmic proteasome, which requires their retrograde transport to the cytoplasm. Based on a co-immunoprecipitation of major histocompatibility complex (MHC) class I heavy-chain breakdown intermediates with the translocon subunit Sec61p, it was speculated that Sec61p maybe involved in retrograde transport. Here we present functional evidence from genetic studies that Sec61p mediates retrograde transport of a mutated lumenal yeast carboxypeptidase ycsY (CPY*) in vivo. The endoplasmic reticulum lumenal chaperone BiP (Kar2p) and Sec63p, which are also subunits of the import machinery, are involved in export of CPY* to the cytosol. Thus our results demonstrate that retrograde transport of proteins is mediated by a functional translocon. We consider the export of endoplasmic reticulum-localized proteins to the cytosol by the translocon for proteasome degradation to be a general process in eukaryotic cell biology.
Processing of NF-kB1 p105 to p50 Restricts Tumor Growth Graphical Abstract Highlights d KPC1 ubiq... more Processing of NF-kB1 p105 to p50 Restricts Tumor Growth Graphical Abstract Highlights d KPC1 ubiquitinates p105, resulting in its basal and signalinduced cleavage to p50 d KPC1 inhibits tumor growth via regulation of p50-dependent tumor suppressor genes d KPC1 and p50 levels are correlated and are lower in tumors than in normal tissue d Excess p50 downregulates p65, possibly lowering tumorigenic levels of p50-p65 NF-kB Authors Yelena Kravtsova-Ivantsiv, Inna Shomer, ..., Ilana Doweck, Aaron Ciechanover Correspondence [email protected] In Brief KPC1-dependent ubiquitination of p105 supports limited processing to produce p50, thereby shaping the population of NF-kB subunits available. Production of p50 appears linked to a tumorsuppressive expression pattern and suggests a balancing of NF-kB's roles in tumorigenesis. Accession Numbers GSE60530 Kravtsova-Ivantsiv et al., 2015, Cell 161, 333-347 April 9, 2015 ª2015 Elsevier Inc. http://dx.
Protein homeostasis is largely dependent on proteolysis by the ubiquitin-proteasome system. Diver... more Protein homeostasis is largely dependent on proteolysis by the ubiquitin-proteasome system. Diverse polyubiquitin modifications are reported to target cellular proteins to the proteasome. At the proteasome, deubiquitination is an essential preprocessing event that contributes to degradation efficiency. We characterized the specificities of two proteasome-associated deubiquitinases (DUBs), Rpn11 and Ubp6, and explored their impact on overall proteasome DUB activity. This was accomplished by constructing a panel of well defined ubiquitin (Ub) conjugates, including homogeneous linkages of varying lengths as well as a heterogeneously modified target. Rpn11 and Ubp6 processed Lys(11) and Lys(63) linkages with comparable efficiencies that increased with chain length. In contrast, processing of Lys(48) linkages by proteasome was inversely correlated to chain length. Fluorescently labeled tetra-Ub chains revealed endo-chain preference for Ubp6 acting on Lys(48) and random action for Rpn11. ...
In cells, both newly synthesized and pre-existing proteins are constantly endangered by misfoldin... more In cells, both newly synthesized and pre-existing proteins are constantly endangered by misfolding and aggregation. The accumulation of damaged proteins can perturb cellular homeostasis and provoke aging, pathological states, and even cell death. To avert these dangers, cells have developed powerful quality control strategies that counteract protein damage in a compartment-specific way. Here, we compare the protein quality control systems of the eukaryotic cytosol and the endoplasmic reticulum, focusing on the principles of damage recognition, the triage decisions between chaperone-mediated refolding and proteolytic elimination of damaged proteins, the repair of misfolded and aggregated protein species, and the mechanisms by which perturbations of protein homeostasis are sensed to induce compartment-specific stress responses.
Cellular quality control mechanisms perform vital tasks by ensuring that the proteome reflects pr... more Cellular quality control mechanisms perform vital tasks by ensuring that the proteome reflects precisely the information encoded by the genome. In this issue of Cell, Gardner et al. (2005) report the discovery of a novel protein quality control system that resides in the nucleus. Central to this system is the E3 ligase San1p, which monitors nuclear proteins and targets aberrant species for destruction.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1997
The endoplasmic reticulum (ER) is the site of entry of proteins into the secretory pathway. It is... more The endoplasmic reticulum (ER) is the site of entry of proteins into the secretory pathway. It is responsible for proper folding of the proteins before delivery to their site of action. Furthermore, proofreading to detect malfolded or unnecessary proteins that have to be eliminated and regulation of protein levels are crucial ER functions. The ubiquitin-proteasome system, located in the cytoplasm, has emerged as the major ER degradation machinery. A multitude of ER resident as well as membrane-bound and soluble proteolytic substrates of the secretory pathway are retained in the ER and destined for degradation via this pathway. Their actual proteolysis is preceded by a retrograde transport to the cytoplasm. A key component of the translocation apparatus, Sec61p, is also the central subunit of the retrograde transport system. Other components of the translocon such as Sec63p or the lumenal chaperone BiP may also be involved in export to the cytosol. Novel ER membrane proteins such as ...
Protein folding within the endoplasmic reticulum (ER) of eukaryotic cells is erroneous and often ... more Protein folding within the endoplasmic reticulum (ER) of eukaryotic cells is erroneous and often results in the formation of terminally malfolded species. A quality control system retards such molecules in the ER and eventually initiates their dislocation into the cytosol for proteolysis by 26S proteasomes. This process is termed ER associated protein degradation (ERAD). The spatial separation of ER based quality control and cytosolic proteolysis poses the need for a machinery that promotes the extraction of substrates from the ER. Due to the heterogeneous nature of the client proteins this transport system displays several unique features. Selective recognition of ERAD substrates does not involve transferable transport signals in the primary sequence and thus must follow other principles than established for proteins designated for the import into organelles. Moreover, an ER dislocation system must be capable to ship polypeptides, which may be at least partly folded and are in most cases covalently modified with bulky and hydrophilic glycans, through a membrane without disrupting the integrity of the ER. In this review we present current ideas on the highly dynamic and flexible nature of the dislocation apparatus and speculate on the mechanism that removes aberrant polypeptides from the ER in the course of ERAD. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.
Proteins damaged by stressors such as heat, oxidizing conditions or toxic agents are deleterious ... more Proteins damaged by stressors such as heat, oxidizing conditions or toxic agents are deleterious to cells and must be properly taken care of. Accordingly, misfolded proteins trigger a cellular stress response that aims to either repair defective polypeptides or eliminate faulty elements when salvage is not possible. This stress response provides time for additional stressor-specific pathways that adapt the cell to the changed environment if necessary. Recent studies have investigated how proteins that frustrate the folding machinery are recognized and cleared from the cell. Surprisingly, these clearance mechanisms are not restricted to the protein level. The stress response can also eliminate the mRNA of polypeptides that are refractory to folding.
In epithelial cells, tyrosine kinases induce the tyrosine phosphorylation and ubiquitination of t... more In epithelial cells, tyrosine kinases induce the tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which induces endocytosis of E-cadherin. With a modified yeast 2-hybrid system, we isolated Hakai, an E-cadherin binding protein, which we have identified as an E3 ubiquitin-ligase. Hakai contains SH2, RING, zinc-finger and proline-rich domains, and interacts with E-cadherin in a tyrosine phosphorylation-dependent manner, inducing ubiquitination of the E-cadherin complex. Expression of Hakai in epithelial cells disrupts cell--cell contacts and enhances endocytosis of E-cadherin and cell motility. Through dynamic recycling of E-cadherin, Hakai can thus modulate cell adhesion, and could participate in the regulation of epithelial--mesenchymal transitions in development or metastasis.
Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), po... more Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. ...
Protein quality control systems protect cells from proteotoxicity caused by the accumulation of a... more Protein quality control systems protect cells from proteotoxicity caused by the accumulation of aberrantly folded polypeptides. The Rsp5 ubiquitin ligase (mammalian homologue Nedd4) is now identified as a major constituent of a clearance pathway that degrades misfolded cytosolic proteins after exposure to heat.
Epitope tagging is a powerful method for the rapid analysis of protein function. In Saccharomyces... more Epitope tagging is a powerful method for the rapid analysis of protein function. In Saccharomyces cerevisiae epitope tags are introduced easily into chromosomal loci by homologous recombination using a simple PCR-based strategy. Although quite a number of tools exist for C-terminal tagging as well as N-terminal tagging of proteins expressed by heterologous promoters, there are only very limited possibilities to tag proteins at the N-terminus and retain the endogenous expression level. Furthermore, no PCR-templates for internal tagging have been reported. Here we describe new modules that are suitable for both the repeated N-terminal and internal tagging of proteins, leaving their endogenous promoters intact. The tags include 6xHA, 9xMyc, yEGFP, TEV-GST-6xHIS, ProtA, TEV-ProtA and TEV-ProtA-7xHIS in conjunction with different heterologous selection markers.
Proteins damaged by stressors such as heat, oxidizing conditions or toxic agents are deleterious ... more Proteins damaged by stressors such as heat, oxidizing conditions or toxic agents are deleterious to cells and must be properly taken care of. Accordingly, misfolded proteins trigger a cellular stress response that aims to either repair defective polypeptides or eliminate faulty elements when salvage is not possible. This stress response provides time for additional stressor-specific pathways that adapt the cell to the changed environment if necessary. Recent studies have investigated how proteins that frustrate the folding machinery are recognized and cleared from the cell. Surprisingly, these clearance mechanisms are not restricted to the protein level. The stress response can also eliminate the mRNA of polypeptides that are refractory to folding.
present in proteins that contain DNAbinding domains, although the PHD finger is found in some pla... more present in proteins that contain DNAbinding domains, although the PHD finger is found in some plant homeodomain proteins 25 . Confirmation of the DNA-binding activity of the DEAF-1 SAND domain would imply that all the SAND proteins are DNA-binding transcription factors. SAND proteins, such as AIRE-1, that also possess PHD fingers are likely to regulate gene expression through the modulation of chromatin structure.
Proteins that fail to fold properly as well as constitutive or regulated short-lived proteins of ... more Proteins that fail to fold properly as well as constitutive or regulated short-lived proteins of the endoplasmic reticulum are subjected to proteolysis by cytosolic 26S proteasomes. This process is known as endoplasmic reticulum-associated protein degradation. In order to become accessible to the proteasome of this system substrates must first be retrogradely transported from the endoplasmic reticulum into the cytosol, in a process termed dislocation. This export step seems to be accompanied by polyubiquitination of such molecules. Surprisingly, protein dislocation from the endoplasmic reticulum seems to require at least some components that mediate import into this compartment. However, protein import and export display differences in the mechanism that provides the driving force and ensures directionality. Of special interest is the cytoplasmic Cdc48p/Npl4p/Ufd1p complex, which is required for the degradation of various endoplasmic reticulum-associated protein degradation substrates and seems to function in a step after polyubiquitination but before proteasomal digestion. In this review, we will summarize our knowledge on protein export during endoplasmic reticulum-associated protein degradation and discuss the possible function of certain components involved in this process.
Endoplasmic reticulum (ER)-associated protein degradation by the ubiquitin-proteasome system requ... more Endoplasmic reticulum (ER)-associated protein degradation by the ubiquitin-proteasome system requires the dislocation of substrates from the ER into the cytosol. It has been speculated that a functional ubiquitin proteasome pathway is not only essential for proteolysis, but also for the preceding export step. Here, we show that short ubiquitin chains synthesized on proteolytic substrates are not sufficient to complete dislocation; the size of the chain seems to be a critical determinant. Moreover, our results suggest that the AAA proteins of the 26S proteasome are not directly involved in substrate export. Instead, a related AAA complex Cdc48, is required for ER-associated protein degradation upstream of the proteasome.
Mistakes are part of our world and constantly occurring. Due to transcriptional and translational... more Mistakes are part of our world and constantly occurring. Due to transcriptional and translational failures, genomic mutations or diverse stress conditions like oxidation or heat misfolded proteins are permanently produced in every compartment of the cell. As misfolded proteins in general lose their native function and tend to aggregate several cellular mechanisms have been evolved dealing with such potentially toxic protein species. Misfolded proteins are mostly recognized by chaperones on the basis of their exposed hydrophobic patches and, if unable to refold them to their native state, are targeted to proteolytic pathways. Most prominent are the ubiquitin-proteasome system and the autophagic vacuolar (lysosomal) system, eliminating misfolded proteins from the cellular environment. A major task of this quality control system is the specific recognition and separation of the misfolded from the correctly folded protein species and the folding intermediates, respectively, which are on the way to the correct folded state but exhibit properties of misfolded proteins. In this review we focus on the recognition process and subsequent degradation of misfolded proteins via the ubiquitin-proteasome system in the different cell compartments of eukaryotic cells. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.
Proteins enter the secretory pathway through the endoplasmic reticulum, which delivers properly f... more Proteins enter the secretory pathway through the endoplasmic reticulum, which delivers properly folded proteins to their site of action and contains a quality-control system to monitor and prevent abnormal proteins from being delivered. Many of these proteins are degraded by the cytoplasmic proteasome, which requires their retrograde transport to the cytoplasm. Based on a co-immunoprecipitation of major histocompatibility complex (MHC) class I heavy-chain breakdown intermediates with the translocon subunit Sec61p, it was speculated that Sec61p maybe involved in retrograde transport. Here we present functional evidence from genetic studies that Sec61p mediates retrograde transport of a mutated lumenal yeast carboxypeptidase ycsY (CPY*) in vivo. The endoplasmic reticulum lumenal chaperone BiP (Kar2p) and Sec63p, which are also subunits of the import machinery, are involved in export of CPY* to the cytosol. Thus our results demonstrate that retrograde transport of proteins is mediated ...
Proteins enter the secretory pathway through the endoplasmic reticulum, which delivers properly f... more Proteins enter the secretory pathway through the endoplasmic reticulum, which delivers properly folded proteins to their site of action and contains a quality-control system to monitor and prevent abnormal proteins from being delivered. Many of these proteins are degraded by the cytoplasmic proteasome, which requires their retrograde transport to the cytoplasm. Based on a co-immunoprecipitation of major histocompatibility complex (MHC) class I heavy-chain breakdown intermediates with the translocon subunit Sec61p, it was speculated that Sec61p maybe involved in retrograde transport. Here we present functional evidence from genetic studies that Sec61p mediates retrograde transport of a mutated lumenal yeast carboxypeptidase ycsY (CPY*) in vivo. The endoplasmic reticulum lumenal chaperone BiP (Kar2p) and Sec63p, which are also subunits of the import machinery, are involved in export of CPY* to the cytosol. Thus our results demonstrate that retrograde transport of proteins is mediated by a functional translocon. We consider the export of endoplasmic reticulum-localized proteins to the cytosol by the translocon for proteasome degradation to be a general process in eukaryotic cell biology.
Processing of NF-kB1 p105 to p50 Restricts Tumor Growth Graphical Abstract Highlights d KPC1 ubiq... more Processing of NF-kB1 p105 to p50 Restricts Tumor Growth Graphical Abstract Highlights d KPC1 ubiquitinates p105, resulting in its basal and signalinduced cleavage to p50 d KPC1 inhibits tumor growth via regulation of p50-dependent tumor suppressor genes d KPC1 and p50 levels are correlated and are lower in tumors than in normal tissue d Excess p50 downregulates p65, possibly lowering tumorigenic levels of p50-p65 NF-kB Authors Yelena Kravtsova-Ivantsiv, Inna Shomer, ..., Ilana Doweck, Aaron Ciechanover Correspondence [email protected] In Brief KPC1-dependent ubiquitination of p105 supports limited processing to produce p50, thereby shaping the population of NF-kB subunits available. Production of p50 appears linked to a tumorsuppressive expression pattern and suggests a balancing of NF-kB's roles in tumorigenesis. Accession Numbers GSE60530 Kravtsova-Ivantsiv et al., 2015, Cell 161, 333-347 April 9, 2015 ª2015 Elsevier Inc. http://dx.
Protein homeostasis is largely dependent on proteolysis by the ubiquitin-proteasome system. Diver... more Protein homeostasis is largely dependent on proteolysis by the ubiquitin-proteasome system. Diverse polyubiquitin modifications are reported to target cellular proteins to the proteasome. At the proteasome, deubiquitination is an essential preprocessing event that contributes to degradation efficiency. We characterized the specificities of two proteasome-associated deubiquitinases (DUBs), Rpn11 and Ubp6, and explored their impact on overall proteasome DUB activity. This was accomplished by constructing a panel of well defined ubiquitin (Ub) conjugates, including homogeneous linkages of varying lengths as well as a heterogeneously modified target. Rpn11 and Ubp6 processed Lys(11) and Lys(63) linkages with comparable efficiencies that increased with chain length. In contrast, processing of Lys(48) linkages by proteasome was inversely correlated to chain length. Fluorescently labeled tetra-Ub chains revealed endo-chain preference for Ubp6 acting on Lys(48) and random action for Rpn11. ...
In cells, both newly synthesized and pre-existing proteins are constantly endangered by misfoldin... more In cells, both newly synthesized and pre-existing proteins are constantly endangered by misfolding and aggregation. The accumulation of damaged proteins can perturb cellular homeostasis and provoke aging, pathological states, and even cell death. To avert these dangers, cells have developed powerful quality control strategies that counteract protein damage in a compartment-specific way. Here, we compare the protein quality control systems of the eukaryotic cytosol and the endoplasmic reticulum, focusing on the principles of damage recognition, the triage decisions between chaperone-mediated refolding and proteolytic elimination of damaged proteins, the repair of misfolded and aggregated protein species, and the mechanisms by which perturbations of protein homeostasis are sensed to induce compartment-specific stress responses.
Cellular quality control mechanisms perform vital tasks by ensuring that the proteome reflects pr... more Cellular quality control mechanisms perform vital tasks by ensuring that the proteome reflects precisely the information encoded by the genome. In this issue of Cell, Gardner et al. (2005) report the discovery of a novel protein quality control system that resides in the nucleus. Central to this system is the E3 ligase San1p, which monitors nuclear proteins and targets aberrant species for destruction.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1997
The endoplasmic reticulum (ER) is the site of entry of proteins into the secretory pathway. It is... more The endoplasmic reticulum (ER) is the site of entry of proteins into the secretory pathway. It is responsible for proper folding of the proteins before delivery to their site of action. Furthermore, proofreading to detect malfolded or unnecessary proteins that have to be eliminated and regulation of protein levels are crucial ER functions. The ubiquitin-proteasome system, located in the cytoplasm, has emerged as the major ER degradation machinery. A multitude of ER resident as well as membrane-bound and soluble proteolytic substrates of the secretory pathway are retained in the ER and destined for degradation via this pathway. Their actual proteolysis is preceded by a retrograde transport to the cytoplasm. A key component of the translocation apparatus, Sec61p, is also the central subunit of the retrograde transport system. Other components of the translocon such as Sec63p or the lumenal chaperone BiP may also be involved in export to the cytosol. Novel ER membrane proteins such as ...
Protein folding within the endoplasmic reticulum (ER) of eukaryotic cells is erroneous and often ... more Protein folding within the endoplasmic reticulum (ER) of eukaryotic cells is erroneous and often results in the formation of terminally malfolded species. A quality control system retards such molecules in the ER and eventually initiates their dislocation into the cytosol for proteolysis by 26S proteasomes. This process is termed ER associated protein degradation (ERAD). The spatial separation of ER based quality control and cytosolic proteolysis poses the need for a machinery that promotes the extraction of substrates from the ER. Due to the heterogeneous nature of the client proteins this transport system displays several unique features. Selective recognition of ERAD substrates does not involve transferable transport signals in the primary sequence and thus must follow other principles than established for proteins designated for the import into organelles. Moreover, an ER dislocation system must be capable to ship polypeptides, which may be at least partly folded and are in most cases covalently modified with bulky and hydrophilic glycans, through a membrane without disrupting the integrity of the ER. In this review we present current ideas on the highly dynamic and flexible nature of the dislocation apparatus and speculate on the mechanism that removes aberrant polypeptides from the ER in the course of ERAD. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.
Proteins damaged by stressors such as heat, oxidizing conditions or toxic agents are deleterious ... more Proteins damaged by stressors such as heat, oxidizing conditions or toxic agents are deleterious to cells and must be properly taken care of. Accordingly, misfolded proteins trigger a cellular stress response that aims to either repair defective polypeptides or eliminate faulty elements when salvage is not possible. This stress response provides time for additional stressor-specific pathways that adapt the cell to the changed environment if necessary. Recent studies have investigated how proteins that frustrate the folding machinery are recognized and cleared from the cell. Surprisingly, these clearance mechanisms are not restricted to the protein level. The stress response can also eliminate the mRNA of polypeptides that are refractory to folding.
In epithelial cells, tyrosine kinases induce the tyrosine phosphorylation and ubiquitination of t... more In epithelial cells, tyrosine kinases induce the tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which induces endocytosis of E-cadherin. With a modified yeast 2-hybrid system, we isolated Hakai, an E-cadherin binding protein, which we have identified as an E3 ubiquitin-ligase. Hakai contains SH2, RING, zinc-finger and proline-rich domains, and interacts with E-cadherin in a tyrosine phosphorylation-dependent manner, inducing ubiquitination of the E-cadherin complex. Expression of Hakai in epithelial cells disrupts cell--cell contacts and enhances endocytosis of E-cadherin and cell motility. Through dynamic recycling of E-cadherin, Hakai can thus modulate cell adhesion, and could participate in the regulation of epithelial--mesenchymal transitions in development or metastasis.
Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), po... more Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. ...
Protein quality control systems protect cells from proteotoxicity caused by the accumulation of a... more Protein quality control systems protect cells from proteotoxicity caused by the accumulation of aberrantly folded polypeptides. The Rsp5 ubiquitin ligase (mammalian homologue Nedd4) is now identified as a major constituent of a clearance pathway that degrades misfolded cytosolic proteins after exposure to heat.
Epitope tagging is a powerful method for the rapid analysis of protein function. In Saccharomyces... more Epitope tagging is a powerful method for the rapid analysis of protein function. In Saccharomyces cerevisiae epitope tags are introduced easily into chromosomal loci by homologous recombination using a simple PCR-based strategy. Although quite a number of tools exist for C-terminal tagging as well as N-terminal tagging of proteins expressed by heterologous promoters, there are only very limited possibilities to tag proteins at the N-terminus and retain the endogenous expression level. Furthermore, no PCR-templates for internal tagging have been reported. Here we describe new modules that are suitable for both the repeated N-terminal and internal tagging of proteins, leaving their endogenous promoters intact. The tags include 6xHA, 9xMyc, yEGFP, TEV-GST-6xHIS, ProtA, TEV-ProtA and TEV-ProtA-7xHIS in conjunction with different heterologous selection markers.
Proteins damaged by stressors such as heat, oxidizing conditions or toxic agents are deleterious ... more Proteins damaged by stressors such as heat, oxidizing conditions or toxic agents are deleterious to cells and must be properly taken care of. Accordingly, misfolded proteins trigger a cellular stress response that aims to either repair defective polypeptides or eliminate faulty elements when salvage is not possible. This stress response provides time for additional stressor-specific pathways that adapt the cell to the changed environment if necessary. Recent studies have investigated how proteins that frustrate the folding machinery are recognized and cleared from the cell. Surprisingly, these clearance mechanisms are not restricted to the protein level. The stress response can also eliminate the mRNA of polypeptides that are refractory to folding.
present in proteins that contain DNAbinding domains, although the PHD finger is found in some pla... more present in proteins that contain DNAbinding domains, although the PHD finger is found in some plant homeodomain proteins 25 . Confirmation of the DNA-binding activity of the DEAF-1 SAND domain would imply that all the SAND proteins are DNA-binding transcription factors. SAND proteins, such as AIRE-1, that also possess PHD fingers are likely to regulate gene expression through the modulation of chromatin structure.
Proteins that fail to fold properly as well as constitutive or regulated short-lived proteins of ... more Proteins that fail to fold properly as well as constitutive or regulated short-lived proteins of the endoplasmic reticulum are subjected to proteolysis by cytosolic 26S proteasomes. This process is known as endoplasmic reticulum-associated protein degradation. In order to become accessible to the proteasome of this system substrates must first be retrogradely transported from the endoplasmic reticulum into the cytosol, in a process termed dislocation. This export step seems to be accompanied by polyubiquitination of such molecules. Surprisingly, protein dislocation from the endoplasmic reticulum seems to require at least some components that mediate import into this compartment. However, protein import and export display differences in the mechanism that provides the driving force and ensures directionality. Of special interest is the cytoplasmic Cdc48p/Npl4p/Ufd1p complex, which is required for the degradation of various endoplasmic reticulum-associated protein degradation substrates and seems to function in a step after polyubiquitination but before proteasomal digestion. In this review, we will summarize our knowledge on protein export during endoplasmic reticulum-associated protein degradation and discuss the possible function of certain components involved in this process.
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