Spatiotemporal protein interactome profiling through condensation-enhanced photocrosslinking

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Protein-protein interactions (PPIs) within biomolecular condensates are vital for cellular functions but challenging to study due to condensate dynamics.
• Existing methods struggle to resolve compartment-specific PPIs within these transient structures.

Purpose of the Study:

• To develop a novel strategy for spatiotemporally resolved mapping of direct protein interactomes within biomolecular condensates.
• To overcome the limitations of studying transient and multi-localized protein interactions within specific cellular compartments.

Main Methods:

• Developed DenseMAP: a condensation-enhanced, spatially directed, metabolic incorporation-assisted photocrosslinking strategy.
• Utilized condensation-enhanced photocrosslinkers and spatially directed biotin tagging for precise interactome mapping.
• Applied DenseMAP to map stress-granule-specific interactomes and nucleolar proteomes.

Main Results:

• Successfully mapped the interactomes of N6-methyladenosine readers YTHDF1 and YTHDF2 within stress granules.
• Uncovered the role of phosphorylation in SARS-CoV-2 nucleocapsid protein in regulating virus replication.
• Deciphered the nucleolar granular component proteome and identified SUMOylation's role in nucleolar homeostasis.

Conclusions:

• DenseMAP is a powerful platform technology for dissecting functional PPI networks within subcellular and subcompartmental condensates.
• The method enables analysis under various physiological and pathological conditions.
• Provides new insights into condensate function, regulation, and disease mechanisms.