Related Experiment Video
Updated: Jun 23, 2025
High-throughput Yeast Plasmid Overexpression Screen
Published on: July 27, 2011
Expression of Wild-Type and Mutant Human TDP-43 in Yeast Inhibits TOROID (TORC1 Organized in Inhibited Domain)
Sangeun Park1, Sei-Kyoung Park1, Susan W Liebman1
1Department of Pharmacology, University of Nevada, Reno, NV 89557, USA.
Abstract:
TDP-43 forms aggregates in the neurons of patients with several neurodegenerative diseases. Human TDP-43 also aggregates and is toxic in yeast. Here, we used a yeast model to investigate (1) the nature of TDP-43 aggregates and (2) the mechanism of TDP-43 toxicity. Thioflavin T, which stains amyloid but not wild-type TDP-43 aggregates, also did not stain mutant TDP-43 aggregates made from TDP-43 with intragenic mutations that increase or decrease its toxicity. However, 1,6-hexanediol, which dissolves liquid droplets, dissolved wild-type or mutant TDP-43 aggregates. To investigate the mechanism of TDP-43 toxicity, the effects of TDP-43 mutations on the autophagy of the GFP-ATG8 reporter were examined. Mutations in TDP-43 that enhance its toxicity, but not mutations that reduce its toxicity, caused a larger reduction in autophagy. TOROID formation, which enhances autophagy, was scored as GFP-TOR1 aggregation. TDP-43 inhibited TOROID formation. TORC1 bound to both toxic and non-toxic TDP-43, and to TDP-43, with reduced toxicity due to pbp1Δ. However, extragenic modifiers and TDP-43 mutants that reduced TDP-43 toxicity, but not TDP-43 mutants that enhanced toxicity, restored TOROID formation. This is consistent with the hypothesis that TDP-43 is toxic in yeast because it reduces TOROID formation, causing the inhibition of autophagy. Whether TDP-43 exerts a similar effect in higher cells remains to be determined.
Insights
TDP-43 protein aggregates in yeast are dissolved by 1,6-hexanediol, not Thioflavin T. Toxic TDP-43 inhibits autophagy by reducing TOROID formation, a key step in the process.
Area of Science:
- Neurobiology
- Cell Biology
- Biochemistry
Background:
- TDP-43 protein forms aggregates in neurons of patients with neurodegenerative diseases.
- TDP-43 aggregation and toxicity are conserved in yeast models.
- Understanding TDP-43 aggregate nature and toxicity mechanisms is crucial for neurodegenerative disease research.
Purpose of the Study:
- To investigate the structural characteristics of TDP-43 aggregates in yeast.
- To elucidate the molecular mechanisms underlying TDP-43-induced toxicity in a yeast model.
- To determine the relationship between TDP-43 aggregation, autophagy, and TOROID formation.
Main Methods:
- Utilized thioflavin T staining and 1,6-hexanediol solubility assays to characterize TDP-43 aggregates.
- Employed a GFP-ATG8 reporter system to assess autophagy modulation by TDP-43.
- Investigated TOROID formation (GFP-TOR1 aggregation) and TORC1 binding to TDP-43.
- Analyzed the impact of TDP-43 mutations and extragenic modifiers on toxicity and autophagy.
Main Results:
- TDP-43 aggregates in yeast were soluble in 1,6-hexanediol but not stained by thioflavin T.
- Mutations enhancing TDP-43 toxicity significantly reduced autophagy, unlike mutations reducing toxicity.
- TDP-43 inhibited TOROID formation, a process essential for autophagy.
- Extragenic modifiers and TDP-43 mutants reducing toxicity restored TOROID formation and autophagy.
Conclusions:
- TDP-43 toxicity in yeast is mediated by the inhibition of TOROID formation, leading to impaired autophagy.
- The findings suggest a conserved mechanism of TDP-43 toxicity involving autophagy disruption.
- Further research is needed to determine if TDP-43 exerts similar effects in higher eukaryotic cells.
Related Concept Videos
01:22PI3K/mTOR/AKT Signaling Pathway
03:03mTOR Signaling and Cancer Progression
The mTOR pathway or the...
01:45Tail-anchoring of Proteins in the ER Membrane