CLPB1/HSP101/HOT1 is the cytosolic/nuclear Arabidopsis Hsp100/ClpB AAA+ chaperone that provides ATP-dependent protein disaggregase activity during heat-stress recovery. Genetic and transgenic studies show that reduced or mutant HSP101 strongly compromises acquired thermotolerance, while domain analysis supports a hexameric threading mechanism that extracts polypeptides from heat-damaged protein aggregates. CLPB1 also supports basal thermotolerance in germinating seeds and, with HSA32, helps maintain long-term acquired thermotolerance through post-transcriptional control of HSA32 and HSP101 protein accumulation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: AtHsp101/CLPB1 is a cytosolic/nuclear protein that functions in the cytoplasm as part of the heat shock response machinery. The protein lacks transit peptides that would direct it to organelles, distinguishing it from organellar ClpB homologs (ClpB3 in chloroplasts, ClpB4 in mitochondria). Reason: This annotation is well-supported by phylogenetic analysis, protein structure (no transit peptides), and functional studies showing cytoplasmic localization and activity. The IBA evidence correctly assigns cytoplasmic localization based on orthology to well-characterized ClpB/Hsp100 proteins. Supporting Evidence: file:ARATH/AT1G74310/AT1G74310-deep-research-perplexity.md AtHsp101/CLPB1 is a cytosolic protein localized to the soluble fraction of the cell, where it functions in the cytoplasm and nucleus as part of the cellular stress response machinery. The protein lacks transit peptides that would direct it to organelles, distinguishing it from the organellar ClpB homologs (ClpB3 in chloroplasts and ClpB4 in mitochondria) that are present in Arabidopsis. |
| GO:0016887 ATP hydrolysis activity | IBA GO_REF:0000033 | ACCEPT | Summary: AtHsp101/CLPB1 is an AAA+ ATPase with two nucleotide-binding domains (NBD1/AAA1 and NBD2/AAA2) that hydrolyze ATP to drive protein disaggregation. ATP hydrolysis is coupled to conformational changes that enable mechanical extraction of polypeptides from aggregates through the central hexameric pore. Reason: This is a core molecular function. The protein contains conserved Walker A, Walker B, and sensor motifs in both NBD1 and NBD2. ATP hydrolysis activity is essential for chaperone function and thermotolerance, as demonstrated by mutants in ATPase domains that lose function. IBA evidence correctly identifies this conserved AAA+ family activity. Supporting Evidence: file:ARATH/AT1G74310/AT1G74310-deep-research-perplexity.md The architecture consists of several critical structural elements an N-terminal domain (NTD), two tandem AAA+ nucleotide-binding domains (designated AAA1/NBD1 and AAA2/NBD2), and a unique coiled-coil middle domain (M-domain) that is inserted between the two nucleotide-binding domains. The first nucleotide-binding domain (NBD1), also termed the large subdomain, contains the Walker A and Walker B motifs essential for ATP binding and hydrolysis. PMID:15659638 The ATPase activity of both NBDs, as well as an intact coiled-coil domain, are essential for chaperone activity and in vivo thermotolerance |
| GO:0034605 cellular response to heat | IBA GO_REF:0000033 | ACCEPT | Summary: AtHsp101/CLPB1 is ESSENTIAL for cellular response to heat stress. The protein mediates acquired thermotolerance and basal thermotolerance through ATP-dependent disaggregation of heat-denatured protein aggregates. Expression increases 50-100 fold within 15-30 minutes of heat stress. Reason: This is the primary biological process for AtHsp101. Multiple lines of evidence demonstrate this is not just "involved in" but ESSENTIAL for heat response. hot1 null mutants completely lack acquired thermotolerance and have dramatically reduced basal thermotolerance. The massive heat-induced transcriptional response and essential role in thermotolerance make this a core annotation. Supporting Evidence: file:ARATH/AT1G74310/AT1G74310-deep-research-perplexity.md Loss-of-function mutations in CLPB1/HSP101 nearly completely abolish this acquired thermotolerance response, rendering hsp101 mutant seedlings sensitive to the same lethal temperature regardless of prior heat conditioning. The transcriptional response of CLPB1/HSP101 to heat stress is among the most robust in the plant transcriptome, with mRNA levels increasing 50- to 100-fold within 15-30 minutes following shift to elevated temperature. file:ARATH/AT1G74310/AT1G74310-notes.md hot1 mutants COMPLETELY LACK acquired thermotolerance. hot1-1 (E637K in NBD2), hot1-3 (null), hot1-4 (A499T in M-domain). Cannot survive 44-45 degrees C even with heat conditioning. |
| GO:0000166 nucleotide binding | IEA GO_REF:0000043 | MODIFY | Summary: Nucleotide binding is a molecular function of AtHsp101 through its two AAA+ domains. However, this term is too general compared to the more specific ATP binding annotation. Reason: While technically correct, this annotation is less specific than GO:0005524 (ATP binding). AtHsp101 specifically binds and hydrolyzes ATP, not other nucleotides. The more specific ATP binding term better captures the molecular function. Proposed replacements: ATP binding Supporting Evidence: file:ARATH/AT1G74310/AT1G74310-deep-research-perplexity.md The first nucleotide-binding domain (NBD1), also termed the large subdomain, contains the Walker A and Walker B motifs essential for ATP binding and hydrolysis, along with conserved sensor residues and an arginine finger motif. The second nucleotide-binding domain (NBD2) similarly contains ATP-binding machinery. |
| GO:0005524 ATP binding | IEA GO_REF:0000120 | ACCEPT | Summary: AtHsp101 contains two ATP-binding domains (NBD1 and NBD2) with conserved Walker A and Walker B motifs for ATP binding. ATP binding is essential for the conformational changes that drive protein disaggregation. Reason: This is a core molecular function that is more specific and accurate than the general nucleotide binding term. Both NBD1 and NBD2 bind ATP specifically, and ATP binding is required for the protein's chaperone activity. The IEA evidence from InterPro domain matches correctly identifies this conserved function. Supporting Evidence: file:ARATH/AT1G74310/AT1G74310-deep-research-perplexity.md Structural analysis demonstrates that ATP binding induces stabilization of the D1 pore loops at the central pore opening, providing a high-affinity substrate-binding state. Upon ATP hydrolysis in the AAA1 ring, conformational changes occur that promote substrate translocation and reduce substrate affinity. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: AtHsp101 localizes to both cytoplasm and nucleus. UniProt annotations indicate nuclear localization, and the protein functions in both compartments as part of the heat shock response machinery. Reason: Nuclear localization is supported by UniProt subcellular location data and consistent with the protein's role in cellular stress response. The cytosolic/nuclear localization distinguishes it from organellar ClpB homologs. Supporting Evidence: file:ARATH/AT1G74310/AT1G74310-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm, cytosol. Nucleus. |
| GO:0005829 cytosol | IEA GO_REF:0000044 | ACCEPT | Summary: AtHsp101 is a cytosolic protein that functions in the soluble fraction of the cell. This is the primary localization where the protein performs its disaggregase activity on heat-denatured protein aggregates. Reason: Cytosol is the primary and most specific subcellular localization for AtHsp101. The protein lacks organellar targeting sequences and functions in the cytosolic compartment. This is more specific than the general cytoplasm annotation and is well-supported by experimental evidence. Supporting Evidence: file:ARATH/AT1G74310/AT1G74310-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm, cytosol. Nucleus. file:ARATH/AT1G74310/AT1G74310-deep-research-perplexity.md AtHsp101/CLPB1 is a cytosolic protein localized to the soluble fraction of the cell, where it functions in the cytoplasm and nucleus as part of the cellular stress response machinery. |
| GO:0009408 response to heat | IEA GO_REF:0000117 | ACCEPT | Summary: AtHsp101 is ESSENTIAL for response to heat stress. The protein is massively induced by heat (50-100 fold increase in mRNA), and hot1 mutants completely lack acquired thermotolerance. This is the defining biological process for this gene. Reason: Response to heat is the primary biological process for AtHsp101. The protein is one of the most heat-inducible genes in Arabidopsis and is absolutely essential for thermotolerance. While GO:0034605 (cellular response to heat) is more specific, this broader term is also accurate and complements the cellular response annotation. Supporting Evidence: file:ARATH/AT1G74310/AT1G74310-notes.md ESSENTIAL for acquired thermotolerance - hot1 mutants completely lack thermotolerance. Massive induction: 50-100 fold increase within 15-30 min of heat stress. PMID:7866032 An Arabidopsis heat shock protein complements a thermotolerance defect in yeast |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | ACCEPT | Summary: This is a duplicate of the IBA annotation for ATP hydrolysis activity (GO:0016887). Both annotations refer to the same core molecular function. Reason: While this is a duplicate annotation with different evidence (IEA from InterPro vs IBA from phylogenetic inference), both correctly identify the same core function. It is acceptable to have the same GO term with different evidence codes, as they provide independent support for the annotation. Supporting Evidence: file:ARATH/AT1G74310/AT1G74310-deep-research-perplexity.md The primary catalytic function of AtHsp101/CLPB1 is to disaggregate heat-denatured proteins that have undergone irreversible aggregation due to thermal stress. This function depends critically on ATP hydrolysis coupled to conformational changes that enable mechanical work. |
| GO:0009644 response to high light intensity | IEP PMID:37947266 Dynamics of mRNA fate during light stress and recovery: from... | KEEP AS NON CORE | Summary: This annotation is based on expression profiling showing AtHsp101 mRNA changes during light stress and recovery. However, this appears to be a secondary or indirect effect rather than a core function of the gene. Reason: The reference (PMID:37947266) focuses on mRNA dynamics during light stress and recovery. AtHsp101 may be induced as part of a general stress response, but light stress is not the primary biological process for this gene. The core function is heat stress response. This annotation represents a pleiotropic effect where heat shock proteins are induced by multiple stresses, but should not be considered a defining feature of AtHsp101 function. Supporting Evidence: PMID:37947266 Dynamics of mRNA fate during light stress and recovery: from transcription to stability and translation. |
| GO:0009570 chloroplast stroma | HDA PMID:20061580 AT_CHLORO, a comprehensive chloroplast proteome database wit... | REMOVE | Summary: This annotation is based on a chloroplast proteomics study (AT_CHLORO database) that detected AtHsp101 in chloroplast fractions. However, this conflicts with the well-established cytosolic/nuclear localization of AtHsp101 (CLPB1) and likely represents contamination or misidentification. Reason: This annotation is inconsistent with extensive evidence that AtHsp101/CLPB1 is a CYTOSOLIC protein that lacks chloroplast transit peptides. Arabidopsis has a distinct chloroplast-targeted ClpB homolog (ClpB3/APG6) that is different from the cytosolic CLPB1. The detection in PMID:20061580 likely represents cytosolic contamination in chloroplast preparations, which is common in organellar proteomics. The protein sequence has no predicted chloroplast targeting signal, and all functional studies demonstrate cytosolic localization. Supporting Evidence: file:ARATH/AT1G74310/AT1G74310-deep-research-perplexity.md AtHsp101/CLPB1 is a cytosolic protein localized to the soluble fraction of the cell. The protein lacks transit peptides that would direct it to organelles, distinguishing it from the organellar ClpB homologs (ClpB3 in chloroplasts and ClpB4 in mitochondria) that are present in Arabidopsis. file:ARATH/AT1G74310/AT1G74310-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm, cytosol. Nucleus. PMID:20061580 Epub 2010 Jan 10. AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins. |
| GO:0009941 chloroplast envelope | HDA PMID:20061580 AT_CHLORO, a comprehensive chloroplast proteome database wit... | REMOVE | Summary: This annotation is from the same chloroplast proteomics study (PMID:20061580) and suffers from the same problem as the chloroplast stroma annotation - likely contamination or misidentification. Reason: AtHsp101/CLPB1 is a cytosolic protein, not a chloroplast protein. The detection in chloroplast envelope fractions is inconsistent with all genetic, biochemical, and sequence evidence showing cytosolic localization. Arabidopsis has a separate chloroplast-targeted ClpB (ClpB3) that is distinct from cytosolic CLPB1. High-throughput proteomics of organellar fractions frequently suffer from cytosolic contamination. Supporting Evidence: file:ARATH/AT1G74310/AT1G74310-deep-research-perplexity.md The identification of distinct organellar and cytosolic ClpB proteins represents an important evolutionary development, as early work on the Arabidopsis ClpB family identified four putative ClpB homologs designated ClpB1-4. The cytosolic AtHsp101 (ClpB1) lacks transit peptides distinguishing it from ClpB3 in chloroplasts and ClpB4 in mitochondria. PMID:20061580 Epub 2010 Jan 10. AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins. |
| GO:0071456 cellular response to hypoxia | HEP PMID:31519798 Integrative Analysis from the Epigenome to Translatome Uncov... | KEEP AS NON CORE | Summary: This annotation is based on high-throughput expression profiling showing changes in AtHsp101 expression during hypoxia stress. However, this represents a secondary stress response rather than a core function. Reason: The HEP (high-throughput expression pattern) evidence indicates AtHsp101 expression changes during hypoxia, but this is not the primary biological process for this gene. Heat shock proteins are often induced by multiple stresses as part of a general stress response. The core function of AtHsp101 is heat stress response and protein disaggregation. Hypoxia response should be considered a pleiotropic or secondary effect. Supporting Evidence: PMID:31519798 Integrative Analysis from the Epigenome to Translatome Uncovers Patterns of Dominant Nuclear Regulation during Transient Stress. |
| GO:0005737 cytoplasm | ISM GO_REF:0000122 | ACCEPT | Summary: This is a duplicate cytoplasm annotation with different evidence (ISM from AtSubP analysis vs IBA from phylogenetic inference). Both correctly identify cytoplasmic localization. Reason: While this duplicates the IBA annotation for cytoplasm, it provides independent computational support (from AtSubP subcellular localization prediction) for the same localization. Having multiple lines of evidence for cytoplasmic localization is acceptable and strengthens the annotation. Supporting Evidence: file:ARATH/AT1G74310/AT1G74310-deep-research-perplexity.md AtHsp101/CLPB1 is a cytosolic protein localized to the soluble fraction of the cell, where it functions in the cytoplasm and nucleus as part of the cellular stress response machinery. |
| GO:0045727 positive regulation of translation | IMP PMID:23439916 Interplay between heat shock proteins HSP101 and HSA32 prolo... | KEEP AS NON CORE | Summary: AtHsp101 supports HSA32 protein accumulation during recovery after heat treatment, but the evidence is narrower than a core translation-recovery role. Reason: PMID:23439916 supports a post-transcriptional HSP101-HSA32 feedback loop that prolongs heat acclimation memory and suggests HSP101 may affect HSA32 synthesis or stability. The paper does not establish stress granule disassembly or broad translation recovery as a primary HSP101 function, so this existing annotation is retained as a non-core, context-specific process. Supporting Evidence: PMID:23439916 Results of immunoblot analyses suggest that HSP101 enhances the translation of HSA32 during recovery after heat treatment, and in turn, HSA32 retards the decay of HSP101. file:ARATH/AT1G74310/AT1G74310-notes.md HSP101 enhances HSA32 translation/accumulation during recovery; HSA32 retards HSP101 decay. This is a feedback loop for long-term heat acclimation memory. |
| GO:0009408 response to heat | IEP PMID:20229063 Functional characterization of AtHsp90.3 in Saccharomyces ce... | ACCEPT | Summary: This is another response to heat annotation with IEP evidence from expression profiling. It duplicates earlier response to heat annotations but with different evidence. Reason: This provides additional experimental support (expression profiling) for the response to heat annotation. Multiple independent studies confirm the heat-responsive nature of AtHsp101. Having the same GO term with different evidence codes and references is acceptable and strengthens the overall annotation. Supporting Evidence: PMID:20229063 Functional characterization of AtHsp90.3 in Saccharomyces cerevisiae and Arabidopsis thaliana under heat stress. |
| GO:0005515 protein binding | IPI PMID:19452453 Proteomic profiling of tandem affinity purified 14-3-3 prote... | MODIFY | Summary: This annotation is based on detection of protein-protein interactions in a 14-3-3 protein complex proteomics study. However, this generic term does not inform about the specific functional interactions. Reason: The supporting paper is a tandem-affinity purification study using 14-3-3 omega (At1g78300) as bait, so the supported interaction is more specifically 14-3-3 protein binding. The generic protein binding term obscures that this is a putative 14-3-3 client interaction and should not be repurposed to annotate CLPB1 substrate binding or Hsp70 cooperation. Proposed replacements: 14-3-3 protein binding Supporting Evidence: PMID:19452453 To uncover new clients, 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a tandem affinity purification tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. file:ARATH/AT1G74310/AT1G74310-goa.tsv The original GOA IPI annotation lists WITH/FROM AGI_LocusCode:At1g78300, the 14-3-3 omega bait used for the interaction evidence. |
| GO:0009408 response to heat | IMP PMID:18047473 The xenobiotic beta-aminobutyric acid enhances Arabidopsis t... | ACCEPT | Summary: Another response to heat annotation with experimental evidence (IMP) from a study on thermotolerance enhancement. This duplicates earlier annotations but provides additional experimental support. Reason: Multiple experimental studies (IMP evidence) independently demonstrate AtHsp101's essential role in heat response. Having the same GO term annotated from multiple independent studies strengthens the overall annotation and reflects the robustness of the experimental evidence. Supporting Evidence: PMID:18047473 The xenobiotic beta-aminobutyric acid enhances Arabidopsis thermotolerance. |
| GO:0009408 response to heat | IMP PMID:15923322 Heat stress phenotypes of Arabidopsis mutants implicate mult... | ACCEPT | Summary: Yet another response to heat annotation with IMP evidence from mutant analysis. This is consistent with all other response to heat annotations. Reason: This study identified heat stress phenotypes of mutants and confirmed AtHsp101's role in thermotolerance acquisition. Multiple IMP annotations from independent studies provide strong experimental support for this core biological process. Supporting Evidence: PMID:15923322 Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. |
| GO:0043335 protein unfolding | IMP PMID:15659638 Genetic analysis reveals domain interactions of Arabidopsis ... | MODIFY | Summary: This annotation describes protein unfolding as a biological process. However, AtHsp101's primary activity is protein disaggregation and refolding, not unfolding per se. Reason: While AtHsp101 does mechanically extract and unfold polypeptides during disaggregation, the term "protein unfolding" can imply the protein's primary role is to unfold native proteins. The better biological-process replacement is protein refolding, because the demonstrated outcome is recovery of aggregated heat-damaged proteins through the Hsp100/ClpB and sHsp/Hsp70 chaperone network. Proposed replacements: protein refolding Supporting Evidence: PMID:15659638 substrate unfolding may be coupled to threading through the axial channel of the ClpB hexamer file:ARATH/AT1G74310/AT1G74310-deep-research-perplexity.md The threading mechanism involves passage of unfolded substrate polypeptides through the central hexameric pore in a processive, directional manner. The actual extraction of individual polypeptides from aggregates involves continuous withdrawal of unfolded protein chains. |
| GO:0009408 response to heat | IEP PMID:7866032 An Arabidopsis heat shock protein complements a thermotolera... | ACCEPT | Summary: The original publication describing AtHsp101 complementation of yeast thermotolerance defects, providing IEP evidence for response to heat. Reason: This is the seminal paper that first characterized AtHsp101 and demonstrated its heat-responsive expression and function in thermotolerance. The IEP evidence from this foundational study is important and complements the other response to heat annotations. Supporting Evidence: PMID:7866032 An Arabidopsis heat shock protein complements a thermotolerance defect in yeast |
| GO:0140545 ATP-dependent protein disaggregase activity | TAS PMID:15659638 Genetic analysis reveals domain interactions of Arabidopsis ... | NEW | Summary: CLPB1/HSP101 is the Arabidopsis cytosolic Hsp100/ClpB AAA+ chaperone whose core molecular activity is ATP-dependent protein disaggregation. Reason: The current annotations include ATP binding and ATP hydrolysis but miss the more informative activity term that captures the Hsp100/ClpB disaggregase function. The Arabidopsis genetic evidence, domain analysis, and conserved ClpB mechanism support ATP-dependent extraction and remodeling of aggregated heat-damaged proteins during thermotolerance recovery. Supporting Evidence: PMID:15659638 In addition, the strongest Class 2 suppressors restored solubility of aggregated small heat shock proteins (sHsps) after heat stress, revealing genetic interaction of the Hsp100/ClpB and sHsp chaperone systems. file:ARATH/AT1G74310/AT1G74310-deep-research-falcon.md The most strongly supported primary biochemical role of Arabidopsis HSP101 is as a protein disaggregase/remodeling chaperone that promotes recovery from heat damage. |
| GO:0042026 protein refolding | TAS PMID:15659638 Genetic analysis reveals domain interactions of Arabidopsis ... | NEW | Summary: AtHsp101 disaggregates heat-denatured protein aggregates, enabling downstream refolding by the cooperating chaperone network. Reason: While protein refolding is not currently annotated, HSP101's ATP-dependent disaggregase activity enables refolding outcomes by extracting or resolubilizing heat-damaged proteins for handling by Hsp70 and related chaperones. The term is justified as the biological outcome of the chaperone network, not as evidence that HSP101 alone performs all refolding steps. Supporting Evidence: PMID:15659638 The strongest Class 2 suppressors restored solubility of aggregated small heat shock proteins (sHsps) after heat stress, revealing genetic interaction of the Hsp100/ClpB and sHsp chaperone systems. file:ARATH/AT1G74310/AT1G74310-notes.md Hierarchical partnership: sHsps (ATP-independent stabilization) → ClpB1 (ATP-dependent disaggregation) → Hsp70 (refolding). |
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