Mutational Scanning of α-Synuclein using a Clickable Protein Tag Reveals Determinants of Membrane-Induced Aggregation

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Cellular environment influences protein conformation and aggregation, which are linked to diseases.
• Studying protein aggregates in living cells at high resolution is challenging.
• Systematic mutagenesis requires robust methods for detecting protein aggregation in numerous variants.

Purpose of the Study:

• To develop a high-throughput method for detecting protein aggregation in living cells.
• To investigate the nature of alpha-synuclein cellular inclusions in a yeast model.
• To identify molecular determinants of membrane-induced protein aggregation.

Main Methods:

• Utilized clickable protein tags to create Förster Resonance Energy Transfer (FRET) pairs in situ.
• Applied high-throughput screening in living yeast cells.
• Performed systematic mutagenesis of alpha-synuclein.

Main Results:

• Demonstrated that cellular alpha-synuclein aggregates in yeast are primarily influenced by protein-membrane interactions.
• Observed that the aggregation pathway in yeast cells differs significantly from in vitro conditions.
• Identified specific residues controlling membrane affinity as key factors in aggregation.
• Discovered glycine residues in the central region act as gatekeepers against membrane-induced aggregation.

Conclusions:

• Clickable protein tags enable high-resolution insights into cellular protein aggregates.
• Protein-membrane interactions are critical in cellular alpha-synuclein aggregation.
• Mutational scanning provides a powerful tool for understanding disease-related protein aggregation mechanisms.