ABSTRACT
Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.
Subject(s)
Adenosine Triphosphatases , DNA Replication , Genomic Instability , Proteostasis , Humans , Adenosine Triphosphatases/metabolism , Valosin Containing Protein/metabolism , Valosin Containing Protein/genetics , HEK293 Cells , Cell Cycle Proteins/metabolism , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/geneticsABSTRACT
Cells must constantly monitor the integrity of their macromolecular constituents. Proteins are the most versatile class of macromolecules but are sensitive to structural alterations. Misfolded or otherwise aberrant protein structures lead to dysfunction and finally aggregation. Their presence is linked to aging and a plethora of severe human diseases. Thus, misfolded proteins have to be rapidly eliminated. Secretory proteins constitute more than one-third of the eukaryotic proteome. They are imported into the endoplasmic reticulum (ER), where they are folded and modified. A highly elaborated machinery controls their folding, recognizes aberrant folding states, and retrotranslocates permanently misfolded proteins from the ER back to the cytosol. In the cytosol, they are degraded by the highly selective ubiquitin-proteasome system. This process of protein quality control followed by proteasomal elimination of the misfolded protein is termed ER-associated degradation (ERAD), and it depends on an intricate interplay between the ER and the cytosol.
Subject(s)
Endoplasmic Reticulum-Associated Degradation , Proteolysis , Saccharomyces cerevisiae Proteins/metabolism , Animals , Cytosol/metabolism , Endoplasmic Reticulum/metabolism , Humans , Models, Biological , Proteasome Endopeptidase Complex/metabolism , Protein Folding , Saccharomyces cerevisiae/metabolism , Ubiquitin/metabolism , Valosin Containing Protein/metabolismABSTRACT
The Cdc48 ATPase and its cofactors Ufd1/Npl4 (UN) extract polyubiquitinated proteins from membranes or macromolecular complexes, but how they perform these functions is unclear. Cdc48 consists of an N-terminal domain that binds UN and two stacked hexameric ATPase rings (D1 and D2) surrounding a central pore. Here, we use purified components to elucidate how the Cdc48 complex processes substrates. After interaction of the polyubiquitin chain with UN, ATP hydrolysis by the D2 ring moves the polypeptide completely through the double ring, generating a pulling force on the substrate and causing its unfolding. ATP hydrolysis by the D1 ring is important for subsequent substrate release from the Cdc48 complex. This release requires cooperation of Cdc48 with a deubiquitinase, which trims polyubiquitin to an oligoubiquitin chain that is then also translocated through the pore. Together, these results lead to a new paradigm for the function of Cdc48 and its mammalian ortholog p97/VCP.
Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/isolation & purification , Amino Acid Sequence , Animals , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/isolation & purification , Endopeptidases/metabolism , Models, Molecular , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin/metabolism , Valosin Containing ProteinABSTRACT
Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex rather than substrate recruitment. Experiments in yeast cells confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.
Subject(s)
Proteasome Endopeptidase Complex , Saccharomyces cerevisiae Proteins , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Polyubiquitin/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin/metabolism , Valosin Containing Protein/genetics , Valosin Containing Protein/metabolismABSTRACT
Misfolded proteins of the ER are retrotranslocated to the cytosol, where they are polyubiquitinated, extracted from the membrane, and degraded by the proteasome. To investigate how the ER-associated Degradation (ERAD) machinery can accomplish retrotranslocation of a misfolded luminal protein domain across a lipid bilayer, we have reconstituted retrotranslocation with purified S. cerevisiae proteins, using proteoliposomes containing the multi-spanning ubiquitin ligase Hrd1. Retrotranslocation of the luminal domain of a membrane-spanning substrate is triggered by autoubiquitination of Hrd1. Substrate ubiquitination is a subsequent event, and the Cdc48 ATPase that completes substrate extraction from the membrane is not required for retrotranslocation. Ubiquitination of lysines in Hrd1's RING-finger domain is required for substrate retrotranslocation in vitro and for ERAD in vivo. Our results suggest that Hrd1 forms a ubiquitin-gated protein-conducting channel.
Subject(s)
Endoplasmic Reticulum-Associated Degradation , Protein Folding , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Ubiquitin-Protein Ligases/metabolism , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Endoplasmic Reticulum/metabolism , Proteolipids/chemistry , Proteolipids/metabolism , Saccharomyces cerevisiae/cytology , Ubiquitination , Valosin Containing ProteinABSTRACT
In this issue of Molecular Cell, Rahmanto et al.1 and Zhao et al.2 demonstrate that RNA-protein crosslinks contribute to formaldehyde toxicity by blocking protein synthesis. Furthermore, they identify a ubiquitin-mediated degradation system for RNA-protein crosslink resolution in eukaryotes.
Subject(s)
Proteasome Endopeptidase Complex , Ubiquitin , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , RNA/genetics , Ubiquitin/metabolism , Valosin Containing Protein/metabolismABSTRACT
The AAA+ ATPase Cdc48 utilizes the cofactor Ufd1/Npl4 to bind and thread polyubiquitinated substrates for their extraction from complexes or membranes and often for subsequent proteasomal degradation. Previous studies indicated that Cdc48 engages polyubiquitin chains through the Npl4-mediated unfolding of an initiator ubiquitin; yet, the underlying principles remain largely unknown. Using FRET-based assays, we revealed the mechanisms and kinetics of ubiquitin unfolding, insertion into the ATPase, and unfolding of the ubiquitin-attached substrate. We found that Cdc48 uses Ufd1's UT3 domain to bind a K48-linked ubiquitin on the initiator's proximal side of the chain, thereby directing the initiator toward rapid unfolding by Npl4 and engagement by Cdc48. Ubiquitins on the initiator's distal side increase substrate affinity and facilitate unfolding but impede substrate release from Cdc48-Ufd1/Npl4 in the absence of additional cofactors. Our findings explain how Cdc48-UN efficiently processes substrates with K48-linked chains of 4-6 ubiquitins, which represent most cellular polyubiquitinated proteins.
Subject(s)
Polyubiquitin , Saccharomyces cerevisiae Proteins , Polyubiquitin/metabolism , Saccharomyces cerevisiae Proteins/metabolism , ATPases Associated with Diverse Cellular Activities/metabolism , Valosin Containing Protein/metabolism , Vesicular Transport Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Ubiquitin/metabolism , Ubiquitins/metabolism , Cell Cycle Proteins/metabolismABSTRACT
Lysosomal membrane permeabilization (LMP) is an underlying feature of diverse conditions including neurodegeneration. Cells respond by extensive ubiquitylation of membrane-associated proteins for clearance of the organelle through lysophagy that is facilitated by the ubiquitin-directed AAA-ATPase VCP/p97. Here, we assessed the ubiquitylated proteome upon acute LMP and uncovered a large diversity of targets and lysophagy regulators. They include calponin-2 (CNN2) that, along with the Arp2/3 complex, translocates to damaged lysosomes and regulates actin filaments to drive phagophore formation. Importantly, CNN2 needs to be ubiquitylated during the process and removed by VCP/p97 for efficient lysophagy. Moreover, we identified the small heat shock protein HSPB1 that assists VCP/p97 in the extraction of CNN2 and show that other membrane regulators including SNAREs, PICALM, AGFG1, and ARL8B are ubiquitylated during lysophagy. Our data reveal a framework of how ubiquitylation and two effectors, VCP/p97 and HSPB1, cooperate to protect cells from the deleterious effects of LMP.
Subject(s)
Macroautophagy , Ubiquitin , Actins/metabolism , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Lysosomes/metabolism , Ubiquitin/metabolism , Valosin Containing Protein/genetics , Valosin Containing Protein/metabolismABSTRACT
The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C terminus through the central pore of the hexameric double ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.
Subject(s)
Polyubiquitin/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Ubiquitinated Proteins/metabolism , Valosin Containing Protein/metabolism , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Transport , Protein Unfolding , Proteolysis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Ubiquitinated Proteins/genetics , Ubiquitination , Valosin Containing Protein/genetics , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolismABSTRACT
Membrane protein clients of endoplasmic reticulum (ER)-associated degradation must be retrotranslocated from the ER membrane by the AAA-ATPase p97 for proteasomal degradation. Before direct engagement with p97, client transmembrane domains (TMDs) that have partially or fully crossed the membrane must be constantly shielded to avoid non-native interactions. How client TMDs are seamlessly escorted from the membrane to p97 is unknown. Here, we identified ER-anchored TMUB1 as a TMD-specific escortase. TMUB1 interacts with the TMD of clients within the membrane and holds â¼10-14 residues of a hydrophobic sequence that is exposed out of membrane, using its transmembrane and cytosolic regions, respectively. The ubiquitin-like domain of TMUB1 recruits p97, which can pull client TMDs from bound TMUB1 into the cytosol. The disruption of TMUB1 escortase activity impairs retrotranslocation and stabilizes retrotranslocating intermediates of client proteins within the ER membrane. Thus, TMUB1 promotes TMD segregation by safeguarding the TMD movement from the membrane to p97.
Subject(s)
Endoplasmic Reticulum , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum-Associated Degradation , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Ubiquitin/metabolism , Valosin Containing Protein/genetics , Valosin Containing Protein/metabolismABSTRACT
Toxic DNA-protein crosslinks (DPCs) arise by ionizing irradiation and UV light, are particularly caused by endogenously produced reactive compounds such as formaldehyde, and also occur during compromised topoisomerase action. Although nucleotide excision repair and homologous recombination contribute to cell survival upon DPCs, hardly anything is known about mechanisms that target the protein component of DPCs directly. Here, we identify the metalloprotease Wss1 as being crucial for cell survival upon exposure to formaldehyde and topoisomerase 1-dependent DNA damage. Yeast mutants lacking Wss1 accumulate DPCs and exhibit gross chromosomal rearrangements. Notably, in vitro assays indicate that substrates such as topoisomerase 1 are processed by the metalloprotease directly and in a DNA-dependent manner. Thus, our data suggest that Wss1 contributes to survival of DPC-harboring cells by acting on DPCs proteolytically. We propose that DPC proteolysis enables repair of these unique lesions via downstream canonical DNA repair pathways.
Subject(s)
DNA Repair , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , DNA/metabolism , DNA Damage , DNA Topoisomerases, Type I/metabolism , Formaldehyde , Sumoylation , Valosin Containing ProteinABSTRACT
Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome, a process called ER-associated protein degradation (ERAD). Here, we use purified components from Saccharomyces cerevisiae to analyze the mechanism of retrotranslocation of luminal substrates (ERAD-L), recapitulating key steps in a basic process in which the ubiquitin ligase Hrd1p is the only required membrane protein. We show that Hrd1p interacts with substrate through its membrane-spanning domain and discriminates misfolded from folded polypeptides. Both Hrd1p and substrate are polyubiquitinated, resulting in the binding of Cdc48p ATPase complex. Subsequently, ATP hydrolysis by Cdc48p releases substrate from Hrd1p. Finally, ubiquitin chains are trimmed by the deubiquitinating enzyme Otu1p, which is recruited and activated by the Cdc48p complex. Cdc48p-dependent membrane extraction of polyubiquitinated proteins can be reproduced with reconstituted proteoliposomes. Our results suggest a model for retrotranslocation in which Hrd1p forms a membrane conduit for misfolded proteins.
Subject(s)
Endoplasmic Reticulum-Associated Degradation , Endoplasmic Reticulum/metabolism , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Endopeptidases/metabolism , Protein Folding , Proteolipids/chemistry , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Valosin Containing ProteinABSTRACT
Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.
Subject(s)
Cullin Proteins/metabolism , Nuclear Proteins/metabolism , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , Transcriptional Elongation Factors/metabolism , Animals , Cell Survival , Chromosomal Proteins, Non-Histone/metabolism , Cullin Proteins/chemistry , Fibroblasts/metabolism , Humans , Indoleacetic Acids/chemistry , Mice , Nedd4 Ubiquitin Protein Ligases/chemistry , Promoter Regions, Genetic , Proteasome Endopeptidase Complex/chemistry , Proteome , Proteomics/methods , Ubiquitin-Protein Ligases/chemistry , Valosin Containing Protein/chemistry , Valosin Containing Protein/metabolismABSTRACT
Stress granules and P bodies are conserved cytoplasmic aggregates of nontranslating messenger ribonucleoprotein complexes (mRNPs) implicated in the regulation of mRNA translation and decay and are related to RNP granules in embryos, neurons, and pathological inclusions in some degenerative diseases. Using baker's yeast, 125 genes were identified in a genetic screen that affected the dynamics of P bodies and/or stress granules. Analyses of such mutants, including CDC48 alleles, provide evidence that stress granules can be targeted to the vacuole by autophagy, in a process termed granulophagy. Moreover, stress granule clearance in mammalian cells is reduced by inhibition of autophagy or by depletion or pathogenic mutations in valosin-containing protein (VCP), the human ortholog of CDC48. Because mutations in VCP predispose humans to amyotrophic lateral sclerosis, frontotemporal lobar degeneration, inclusion body myopathy, and multisystem proteinopathy, this work suggests that autophagic clearance of stress granule related and pathogenic RNP granules that arise in degenerative diseases may be important in reducing their pathology.
Subject(s)
Adenosine Triphosphatases/metabolism , Autophagy , Cell Cycle Proteins/metabolism , Cytoplasmic Granules/metabolism , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphatases/genetics , Animals , Cell Cycle Proteins/genetics , Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibroblasts/metabolism , HeLa Cells , Humans , Mice , Mutation , RNA Stability , RNA, Messenger/metabolism , Ribonucleoproteins/genetics , Saccharomyces cerevisiae Proteins , Valosin Containing ProteinABSTRACT
Valosin-containing protein (VCP)/p97 is an AAA-ATPase that extracts polyubiquitinated substrates from multimeric macromolecular complexes and biological membranes for proteasomal degradation. During p97-mediated extraction, the substrate is largely deubiquitinated as it is threaded through the p97 central pore. How p97-extracted substrates are targeted to the proteasome with few or no ubiquitins is unknown. Here, we report that p97-extracted membrane proteins undergo a second round of ubiquitination catalyzed by the cytosolic ubiquitin ligase RNF126. RNF126 interacts with transmembrane-domain-specific chaperone BAG6, which captures p97-liberated substrates. RNF126 depletion in cells diminishes the ubiquitination of extracted membrane proteins, slows down their turnover, and dramatically stabilizes otherwise transient intermediates in the cytosol. We reconstitute the reubiquitination of a p97-extracted, misfolded multispanning membrane protein with purified factors. Our results demonstrate that p97-extracted substrates need to rapidly engage ubiquitin ligase-chaperone pairs that rebuild the ubiquitin signal for proteasome targeting to prevent harmful accumulation of unfolded intermediates.
Subject(s)
Membrane Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Ubiquitin-Protein Ligases/metabolism , Valosin Containing Protein/metabolism , Catalysis , Cytosol/metabolism , HEK293 Cells , Humans , Molecular Chaperones/metabolism , Protein Folding , Proteolysis , Solubility , UbiquitinationABSTRACT
The ubiquitin ligase Parkin, protein kinase PINK1, USP30 deubiquitylase, and p97 segregase function together to regulate turnover of damaged mitochondria via mitophagy, but our mechanistic understanding in neurons is limited. Here, we combine induced neurons (iNeurons) derived from embryonic stem cells with quantitative proteomics to reveal the dynamics and specificity of Parkin-dependent ubiquitylation under endogenous expression conditions. Targets showing elevated ubiquitylation in USP30-/- iNeurons are concentrated in components of the mitochondrial translocon, and the ubiquitylation kinetics of the vast majority of Parkin targets are unaffected, correlating with a modest kinetic acceleration in accumulation of pS65-Ub and mitophagic flux upon mitochondrial depolarization without USP30. Basally, ubiquitylated translocon import substrates accumulate, suggesting a quality control function for USP30. p97 was dispensable for Parkin ligase activity in iNeurons. This work provides an unprecedented quantitative landscape of the Parkin-modified ubiquitylome in iNeurons and reveals the underlying specificity of central regulatory elements in the pathway.
Subject(s)
Human Embryonic Stem Cells/enzymology , Mitochondria/enzymology , Mitochondrial Proteins/metabolism , Mitophagy , Neural Stem Cells/enzymology , Neurogenesis , Neurons/enzymology , Thiolester Hydrolases/metabolism , Ubiquitin-Protein Ligases/metabolism , HeLa Cells , Human Embryonic Stem Cells/pathology , Humans , Kinetics , Mitochondria/genetics , Mitochondria/pathology , Mitochondrial Proteins/genetics , Neural Stem Cells/pathology , Neurons/pathology , Phosphorylation , Protein Kinases/genetics , Protein Kinases/metabolism , Proteomics , Signal Transduction , Thiolester Hydrolases/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitination , Valosin Containing Protein/genetics , Valosin Containing Protein/metabolismABSTRACT
The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is unclear. Here, we present cryo-EM structures of p97 in the process of disassembling a protein phosphatase-1 (PP1) complex by extracting an inhibitory subunit from PP1. We show that PP1 and its partners SDS22 and inhibitor-3 (I3) are loaded tightly onto p97, surprisingly via a direct contact of SDS22 with the p97 N-domain. Loading is assisted by the p37 adapter that bridges two adjacent p97 N-domains underneath the substrate complex. A stretch of I3 is threaded into the central channel of the spiral-shaped p97 hexamer, while other elements of I3 are still attached to PP1. Thus, our data show how p97 arranges a protein complex between the p97 N-domain and central channel, suggesting a hold-and-extract mechanism for p97-mediated disassembly.
Subject(s)
Cell Cycle Proteins , Ubiquitin , Ubiquitin/metabolism , Protein Phosphatase 1/genetics , Protein Phosphatase 1/metabolism , ATPases Associated with Diverse Cellular Activities/metabolism , Models, Molecular , Valosin Containing Protein/genetics , Valosin Containing Protein/metabolism , Cell Cycle Proteins/metabolismABSTRACT
The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.
Subject(s)
Multiprotein Complexes/metabolism , Protein Biosynthesis , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/genetics , Heat-Shock Proteins/genetics , Peptides/metabolism , Proteasome Endopeptidase Complex/metabolism , RNA-Binding Proteins , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Stress, Physiological , Transcription Factors/genetics , Ubiquitin-Protein Ligases/metabolism , Valosin Containing ProteinABSTRACT
Normal mitochondrial functions rely on optimized composition of their resident proteins, and proteins mistargeted to mitochondria need to be efficiently removed. Msp1, an AAA-ATPase in the mitochondrial outer membrane (OM), facilitates degradation of tail-anchored (TA) proteins mistargeted to the OM, yet how Msp1 cooperates with other factors to conduct this process was unclear. Here, we show that Msp1 recognizes substrate TA proteins and facilitates their transfer to the endoplasmic reticulum (ER). Doa10 in the ER membrane then ubiquitinates them with Ubc6 and Ubc7. Ubiquitinated substrates are extracted from the ER membrane by another AAA-ATPase in the cytosol, Cdc48, with Ufd1 and Npl4 for proteasomal degradation in the cytosol. Thus, Msp1 functions as an extractase that mediates clearance of mistargeted TA proteins by facilitating their transfer to the ER for protein quality control.
Subject(s)
Adenosine Triphosphatases/metabolism , Endoplasmic Reticulum/enzymology , Mitochondria/enzymology , Mitochondrial Membranes/enzymology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Adenosine Triphosphatases/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Transport , Proteolysis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Valosin Containing Protein/genetics , Valosin Containing Protein/metabolismABSTRACT
Similar to ubiquitin, SUMO forms chains, but the identity of SUMO-chain-modified factors and the purpose of this modification remain largely unknown. Here, we identify the budding yeast SUMO protease Ulp2, able to disassemble SUMO chains, as a DDK interactor enriched at replication origins that promotes DNA replication initiation. Replication-engaged DDK is SUMOylated on chromatin, becoming a degradation-prone substrate when Ulp2 no longer protects it against SUMO chain assembly. Specifically, SUMO chains channel DDK for SUMO-targeted ubiquitin ligase Slx5/Slx8-mediated and Cdc48 segregase-assisted proteasomal degradation. Importantly, the SUMOylation-defective ddk-KR mutant rescues inefficient replication onset and MCM activation in cells lacking Ulp2, suggesting that SUMO chains time DDK degradation. Using two unbiased proteomic approaches, we further identify subunits of the MCM helicase and other factors as SUMO-chain-modified degradation-prone substrates of Ulp2 and Slx5/Slx8. We thus propose SUMO-chain/Ulp2-protease-regulated proteasomal degradation as a mechanism that times the availability of functionally engaged SUMO-modified protein pools during replication and beyond.