HSP104 Saccharomyces

What is the primary function of HSP104 in Saccharomyces cerevisiae?

HSP104 functions as a stress-responsive molecular chaperone that resolubilizes protein aggregates formed under stress conditions, particularly heat stress. It belongs to the AAA+ (ATPases Associated with diverse cellular Activities) protein family and utilizes ATP hydrolysis to extract and unfold proteins from aggregates. In yeast cells, HSP104 serves a crucial role in thermotolerance and plays a unique function in the propagation of certain prions, most notably the [PSI+] prion (formed by aggregated Sup35 protein). During normal cellular function, HSP104 maintains proteostasis by resolubilizing stress-damaged proteins, while also facilitating prion propagation through its severing activity that creates new propagons from larger amyloid fibers .

How is HSP104 expression regulated under different conditions?

HSP104 expression is maintained at basal levels under normal growth conditions but is dramatically induced in response to various stresses. Western blot analysis demonstrates that exposure to 10% ethanol increases HSP104 expression approximately 3-fold compared to control conditions . Similarly, heat shock treatment significantly upregulates HSP104, as evidenced by increased HSP104GFP fluorescence . The HSP104 gene contains heat shock elements (HSEs) in its promoter region that are bound by heat shock transcription factors (HSFs) upon stress activation. Carbon source also influences HSP104 expression, with differential expression observed between glucose and galactose-based media . These regulatory mechanisms ensure HSP104 availability matches cellular stress demands.

What distinguishes weak and strong [PSI+] variants in their response to HSP104?

[PSI+] prion exists in different variants (or strains) with distinct conformations and biophysical properties. Weak [PSI+] variants contain fewer seeds and more soluble Sup35 compared to strong [PSI+] variants . Experimental evidence demonstrates these variants respond differently to HSP104 manipulation: weak [PSI+] variants are cured much faster by HSP104 overexpression than strong variants. The L1758 weak [PSI+] strain shows approximately 50% curing per generation with HSP104 overexpression, compared to only about 15% curing per generation observed with stronger [PSI+] variants . Furthermore, even low levels of HSP104 overexpression can cure weak [PSI+] variants, while strong [PSI+] variants require higher HSP104 levels for effective curing . These differential responses reflect underlying structural differences between prion variants.

What techniques effectively measure HSP104 expression and activity?

Several complementary approaches can effectively measure HSP104 expression and activity:

• Western blotting: The gold standard for quantifying HSP104 protein levels, typically normalized to loading controls such as PGK1. This approach revealed a ~3-fold increase in HSP104 levels in both ethanol-treated cells and cells overexpressing HSP104 from the GAL1 promoter .

• Fluorescence microscopy: Using HSP104GFP fusion proteins allows visualization of HSP104 localization and relative expression levels. This technique has demonstrated increased HSP104 fluorescence following heat shock treatment .

• Red/white colony assay: This functional assay measures HSP104's activity in [PSI+] prion curing. [PSI+] cells form white or pink colonies, while cured cells ([psi-]) form red colonies. Complete curing is indicated by fully red colonies rather than red/white sectored colonies .

• Growth recovery assays: Measuring yeast viability and growth resumption after stress treatments indirectly assesses HSP104's ability to help cells recover from stress-induced protein aggregation .

• Stress sensitivity assays: Comparing wildtype, mutant, and ∆hsp104 cells' sensitivity to stressors like DTT, H2O2, and high salt provides functional insights into HSP104 activity .

How can researchers effectively study HSP104-prion interactions?

To effectively study HSP104-prion interactions, researchers can employ multiple complementary approaches:

• Curing assays: Induce HSP104 overexpression (e.g., from the GAL1 promoter) and measure prion curing rates using the red/white colony assay . This approach allows comparison of different prion variants or testing effects of HSP104 mutants.

• Mother-daughter cell separation: Separate mother and daughter cells after inducing HSP104 overexpression to determine differences in curing rates. This can be accomplished by labeling yeast cell walls with fluorescent tags before HSP104 induction, then separating fluorescent mother cells from non-fluorescent daughter cells via flow cytometry .

• Cell division inhibition: Inhibit cell division using hydroxyurea or ethanol while overexpressing HSP104 to distinguish between curing mechanisms requiring or independent of cell division .

• Imaging of prion aggregates: Track GFP-labeled Sup35 foci to observe dissolution of prion aggregates during HSP104 overexpression . This provides direct visual evidence of HSP104's effect on prion structures.

• Genetic approaches: Test requirements for specific genes (like SIR2) in HSP104-mediated prion curing through deletion studies . This helps identify cellular factors involved in the curing process.

What controls are critical when studying HSP104-mediated prion curing?

Robust controls are essential when studying HSP104-mediated prion curing to avoid misinterpretation:

• Strain controls: Include both [PSI+] and [psi-] controls to validate prion-based assays. Different [PSI+] variants respond differently to HSP104, so strain selection critically impacts results .

• Expression verification: Use Western blots with appropriate loading controls (like PGK1) to verify HSP104 expression levels . The degree of overexpression directly influences curing efficiency.

• Growth measurement: Monitor cell growth (via absorbance at 600nm) alongside curing to account for cell division effects . This distinguishes between curing mechanisms dependent on or independent of cell division.

• Viability controls: When using treatments that might affect cell viability (like ethanol), test viability by plating cells on YPD plates before and after treatment . This ensures observed effects aren't due to cell death.

• Vector controls: Include the same strain transformed with an empty vector when overexpressing HSP104 . This controls for effects of the transformation process.

• Time course measurements: Measure curing at multiple time points to capture process kinetics . Different mechanisms of curing show distinct kinetic profiles.

How do HSP104's severing and trimming activities mechanistically differ?

HSP104 exhibits two distinct activities on prion fibers that have different functional outcomes:

• Severing activity: Fragments prion fibers into smaller pieces, increasing the number of propagation units (seeds) while decreasing their average size . This activity predominates at normal HSP104 expression levels and promotes prion propagation by creating new seeds that can be transmitted to daughter cells.

• Trimming activity: Makes seeds smaller without changing their number . This activity becomes more dominant during HSP104 overexpression and leads to the dissolution of prion seeds when they become too small to maintain their amyloid structure.

These activities likely reflect different HSP104 binding orientations to substrates, different configurations within the HSP104 hexamer, varied patterns of ATP hydrolysis, or distinct interactions with co-chaperones. The mechanistic balance between these activities explains how HSP104 can both maintain and cure prions depending on its expression level .

What evidence supports the dissolution model versus asymmetric segregation model?

Multiple experimental lines strongly support the dissolution model over the asymmetric segregation model for HSP104-mediated [PSI+] curing:

These findings collectively provide compelling evidence for the dissolution model, where HSP104 overexpression directly dissolves prion seeds through enhanced trimming activity.

How does ethanol affect [PSI+] curing independently of plasmid-driven HSP104 overexpression?

Ethanol exerts a significant effect on [PSI+] curing independent of plasmid-driven HSP104 overexpression through several mechanisms:

• HSP104 induction: Western blot analysis demonstrates that 10% ethanol induces approximately 3-fold higher HSP104 expression from the endogenous promoter, comparable to levels observed with the GAL1 promoter during early induction . This increased expression is sufficient to cure weak [PSI+] variants.

• Cell division inhibition: Ethanol arrests cell growth for approximately 6 hours, with only 0.3 generations of growth occurring during 4 hours in 10% ethanol . This eliminates dilution effects and allows the direct dissolution mechanism to operate more efficiently.

• Effective curing without plasmid overexpression: When the L1758 [PSI+] strain transformed with an empty vector was exposed to 10% ethanol, approximately 85% of cells were cured after 4 hours despite growing only 0.3 generations . This demonstrates ethanol alone is sufficient to cure [PSI+].

• Carbon source interactions: The curing effect of ethanol occurs with different fermentable carbon sources, though the efficiency varies. After 4 hours in 10% ethanol, approximately 50% of L1758 [PSI+] cells were cured in synthetic medium with glucose .

These findings have significant implications for understanding both HSP104 regulation and prion biology, demonstrating that natural stress conditions can induce sufficient HSP104 levels to cure certain prion variants.

How does HSP104 respond to different types of cellular stress?

HSP104 responds differentially to various stressors, reflecting its adaptable role in maintaining proteostasis:

• Heat stress: Heat shock significantly increases HSP104GFP fluorescence, indicating upregulated expression . Heat shock also affects HSP104 aggregation patterns, with more aggregation observed in wildtype HSP104 than mutant forms after heat shock in aged cells .

• Ethanol stress: Exposure to 10% ethanol increases HSP104 expression approximately 3-fold compared to control conditions . Ethanol simultaneously induces HSP104 expression and arrests cell division, creating ideal conditions for studying division-independent HSP104 functions .

• Oxidative stress: ∆hsp104 cells show higher sensitivity to H₂O₂ than wildtype and mutant cells, indicating HSP104 contributes substantially to oxidative stress resistance . This suggests HSP104 resolubilizes proteins damaged by oxidation.

• Reducing stress: ∆hsp104 cells demonstrate increased sensitivity to dithiothreitol (DTT) compared to wildtype and mutant cells . DTT causes protein misfolding by disrupting disulfide bonds, suggesting HSP104 helps manage this form of proteotoxic stress.

• Osmotic stress: Interestingly, HSP104 wildtype cells show higher sensitivity to high salt conditions than HSP104 mutant and ∆hsp104 cells . This suggests a more complex relationship between HSP104 and osmotic stress responses that may involve regulatory interactions with other cellular systems.

These differential responses indicate HSP104 plays distinct roles across stress conditions, likely through interactions with stress-specific substrates or co-chaperones.

How does HSP104 function change with cellular age?

HSP104 function undergoes significant changes with cellular aging:

• Increased aggregation propensity: Experimental data indicates HSP104 protein aggregation increases with cellular age, with more aggregation observed in HSP104 wildtype than mutant forms after heat shock in aged cells . This suggests HSP104 itself becomes less soluble during aging.

• Altered subcellular localization: HSP104 shows age-dependent changes in localization patterns, raising questions about what cellular events trigger this redistribution . This altered localization may affect HSP104's ability to interact with substrates or co-chaperones.

• Stress response interactions: The relationship between aging and stress responses appears complex, as research has focused on whether HSP104's age-dependent subcellular accumulation can be counteracted or reinforced by various stressors .

• Functional implications: These age-related changes likely affect HSP104's ability to maintain proteostasis and manage protein aggregation in older cells. Declining HSP104 function may contribute to the accumulated protein damage observed in aged cells.

Understanding these age-dependent changes has broader implications for protein quality control during aging. Determining whether HSP104's diminished function is a cause or consequence of aging could provide insights into fundamental aging mechanisms and potential interventions to maintain proteostasis in older cells.

What are the key methodological considerations for HSP104 mutant analysis?

When analyzing HSP104 mutants, researchers should implement these methodological approaches:

• Expression level verification: Quantify mutant expression levels via Western blotting with appropriate loading controls. Differences in expression can confound functional comparisons between wildtype and mutant proteins .

• Protein stability assessment: Evaluate protein stability using cycloheximide chase experiments, which revealed greater stability in HSP104 wildtype compared to mutant forms . This approach distinguishes between functional defects and reduced protein levels.

• Stress response profiling: Compare responses to diverse stressors (heat, oxidative, reducing, osmotic) between wildtype, mutant, and deletion strains. These comprehensive stress profiles revealed that ∆hsp104 cells show higher sensitivity to DTT and H₂O₂ than wildtype and mutant cells, while HSP104 wildtype cells show greater sensitivity to high salt oxidative stress than mutant or deletion strains .

• Age-dependent analysis: Evaluate how mutant phenotypes change with cellular age. HSP104 protein aggregation increases with age, with differences between wildtype and mutant forms becoming more pronounced in older cells .

• Fluorescence microscopy: Use fluorescently tagged proteins to track localization patterns. HSP104GFP showed increased fluorescence in both wildtype and mutant forms after heat shock, providing insights into stress-responsive expression and localization .

These methodological considerations ensure robust characterization of HSP104 mutants and facilitate meaningful comparisons that advance our understanding of structure-function relationships.

What are the unresolved questions about HSP104's role in different [PSI+] variants?

Despite significant progress, several important questions about HSP104's interaction with different [PSI+] variants remain unresolved:

• Structural basis for variant sensitivity: While weak [PSI+] variants are more easily cured by HSP104 overexpression than strong variants , the structural features determining this differential sensitivity remain incompletely understood. Identifying specific conformational elements that influence HSP104 accessibility or activity would provide mechanistic insights.

• Variant-specific interaction sites: The precise binding interfaces between HSP104 and different [PSI+] variants have not been fully mapped. Understanding these interaction sites could explain variant-specific responses and potentially enable targeted interventions.

• Co-chaperone requirements: Different prion variants may require distinct co-chaperone factors for propagation or curing. The search results mention that proteins like Hsp90 and STI1 affect [PSI+] curing rates by HSP104 overexpression , but variant-specific co-chaperone requirements remain unclear.

• Curing kinetics mechanisms: While weak variants cure faster than strong variants, the molecular basis for these kinetic differences is not fully elucidated. The relationship between prion structure, HSP104 binding affinity, processing efficiency, and curing rates requires further investigation.

• Variant formation and selection: The role of HSP104 in the initial formation of different [PSI+] variants and potential selection pressures that influence variant predominance in different conditions remains an active research area.

Resolving these questions would enhance our understanding of both HSP104 function and prion biology while potentially informing approaches to manage protein misfolding diseases in higher organisms.