Systematic Protein-Protein Interaction Analysis Reveals Intersubcomplex Contacts in the Nuclear Pore Complex

Area of Science:

• Cell Biology
• Structural Biology
• Biochemistry

Background:

• The nuclear pore complex (NPC) regulates transport between the nucleus and cytoplasm.
• NPCs are massive protein assemblies (∼40-112 MDa) composed of nucleoporins (Nups).
• Determining NPC structure and assembly is challenging due to its size and complexity.

Purpose of the Study:

• To systematically map direct protein-protein interactions between nucleoporins (Nups).
• To provide experimental data for understanding NPC structural organization and assembly pathways.
• To investigate potential variations in the structural context of major NPC subcomplexes.

Main Methods:

• Comprehensive yeast two-hybrid (Y2H) interaction matrix screening in yeast and human cells.
• Benchmarking Y2H data against existing crystallographic and co-affinity purification data.
• Biophysical validation using microscale thermophoresis and protein fragment complementation assays.

Main Results:

• A high-coverage, accurate map of direct Nup-Nup interactions was generated.
• Novel inter-subcomplex interactions within the NPC were identified and validated.
• Data supports a model where major NPC architectural elements, like the Y-complex, adopt different structures.

Conclusions:

• The generated Nup interaction map offers insights into NPC structural organization.
• Findings provide evidence for dynamic structural arrangements of NPC components during assembly.
• The study favors an NPC assembly model involving context-dependent structures of key subcomplexes.