PAINT/IBA methodology propagates experimental annotations within a phylogenetic tree. Source of all 8 IBA annotations on HSP104: cytoplasm (GO:0005737), ATP hydrolysis activity (GO:0016887), protein refolding (GO:0042026), protein unfolding (GO:0043335), cytosol (GO:0005829), unfolded protein binding (GO:0051082), protein-folding chaperone binding (GO:0051087), and cellular heat acclimation (GO:0070370). All are consistent with the well-established HSP100/Clp disaggregase family function.

UniProt keyword-to-GO mapping methodology. Source of the IEA "nucleotide binding" (GO:0000166) annotation for HSP104, derived from a Swiss-Prot keyword and consistent with the AAA+ ATPase architecture of the protein.

UniProt subcellular-location-to-GO mapping methodology. Source of the IEA "nucleus" (GO:0005634) and "cytoplasm" (GO:0005737) annotations for HSP104, both consistent with the experimentally established localization of the disaggregase (PMID:10467108).

ARBA machine-learning rule-mining methodology that propagates GO terms from UniProtKB feature evidence. Source of six IEA annotations on HSP104: cytosol (GO:0005829), protein folding (GO:0006457), cellular response to heat (GO:0034605), identical protein binding (GO:0042802), protein unfolding (GO:0043335), and intracellular organelle lumen (GO:0070013).

Combined automated annotation pipeline that aggregates multiple electronic GO assignment methods. Source of the IEA "ATP binding" (GO:0005524) and "ATP hydrolysis activity" (GO:0016887) annotations on HSP104, which are redundant with experimental evidence for the ATP-dependent disaggregase activity.

HSP104 deletion mutants cannot acquire thermotolerance after mild heat pretreatment, establishing HSP104 as essential for induced thermotolerance.

HSP104 mediates resolubilization of heat-inactivated protein from insoluble aggregates, establishing the disaggregation function.

HSP104 in concert with Hsp70 and Hsp40 can reactivate aggregated proteins via a protein remodeling activity requiring specific chaperone interactions.

HSP104 disruption affects trehalose metabolism during heat shock, likely through indirect effects on enzyme integrity.

ImmunoEM shows HSP104 in cytoplasm and nucleus, concentrating around protein aggregates during heat shock.

Cytosolic HSP104 is required for conformational repair of heat-denatured proteins in the ER, suggesting indirect role in ER protein quality control.

Fluorescent probe (Y819W) directly measures ATP and ADP binding at NBD2; R826M sensor-2 mutation decreases nucleotide affinity and reveals interdomain communication.

ATP-dependent substrate binding demonstrated; ATP at NBD1 is crucial for polypeptide interaction, and substrate binding triggers conformational change committing to ATP hydrolysis.

High-throughput TAP-tag/MS study identifying HSP104 interactions with multiple yeast proteins.

Systematic TAP-tag analysis of all 63 yeast chaperones, mapping HSP104 interaction network.

CryoEM structure confirms hexameric ring architecture with M-domain on the exterior, where it can interact with aggregated proteins.

HSP104 identified as part of the TRC complex for tail-anchored protein targeting to ER.

Middle domain of HSP104 determines species-specific interaction with Hsp70, required for disaggregation and thermotolerance.

High-throughput microscopy confirms HSP104 cytoplasmic localization.

HSP104 and Hsp70 are required for stress granule disassembly and restoration of translation during heat stress recovery.

HSP104 localizes at the intranuclear quality control compartment (INQ) at the nuclear periphery.

Substrate threading through HSP104's central pore is the mechanism for both protein disaggregation and prion propagation.

Hsp104 is a hexameric AAA+ ATP-dependent disaggregase of the Hsp100/ClpB family that does not perform proteolysis but instead remodels aggregated proteins to enable their reactivation; together with partner chaperones it rescues previously aggregated proteins rather than merely preventing aggregation.

Hsp104 expression is sufficient for acquired thermotolerance and promotes resolubilization and reactivation of proteins that unfolded and aggregated after heat shock.

Hsp104 is a hexameric AAA+ ATPase with two nucleotide-binding domains (NBD1 and NBD2); it extracts clients by ATP-driven unfolding and threading through an axial channel, with the NBD2 pore-loop (conserved GYVG-loop, Tyr-662) required for disaggregation.

Hsp104 operates in a multichaperone disaggregation pathway with Hsp70 and Hsp40; reactivation of aggregated luciferase requires ATP and depends on SSA-encoded Hsp70 and the Hsp40 co-chaperone Ydj1.

Hsp110 nucleotide-exchange factors (Sse1/Sse2) coordinate Hsp70 cycling and are essential for efficient Hsp104-dependent disaggregation in both cytosol and nucleus.

A defining yeast-specific role of Hsp104 is amyloid prion fibril fragmentation, generating transmissible seeds (propagons) needed for prion inheritance such as [PSI+].

Hsp104 levels must be finely tuned: both deletion and overexpression can cure [PSI+], and Hsp70 (Ssa1) antagonizes Hsp104 overexpression-mediated curing.

Hsp104 is predominantly cytosolic under non-stress conditions but relocalizes to stress-induced protein quality control foci associated with stress granules, Q-bodies/CytoQ, and nuclear deposits (INQ/IPOD).

Under glucose starvation, ATP depletion drives sequestration of proteins into stress compartments (stress granules and Q-bodies), and Hsp104 ATPase activity is a key ATP-consuming process governing the abundance and size of these compartments.

Heat-induced stress granules (Pab1-GFP) resolve within ~2 h in wild-type cells but recovery is dramatically slower in hsp104-delta cells, consistent with an Hsp104-dependent clearance process.

Nucleotide-state-specific middle-domain (MD):NBD contacts tune Hsp104 activity and Hsp70 collaboration, and the MD can help ensure that processed substrates remain functional after Hsp104 action.