LAP2 binds to BAF.DNA complexes: requirement for the LEM domain and modulation by variable regions

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Lamina-associated polypeptide 2 (LAP2) proteins are integral nuclear membrane proteins characterized by a conserved LEM domain.
• LAP2 isoforms share a common N-terminal region (LAP2-c), including the LEM domain, and possess variable C-terminal regions.
• LAP2-c inhibits nuclear assembly in Xenopus egg extracts and binds to the DNA-bridging protein, barrier-to-autointegration factor (BAF).

Purpose of the Study:

• To investigate the functional domains of Xenopus LAP2-c responsible for nuclear assembly inhibition and BAF binding.
• To elucidate the role of the LEM domain and other regions in LAP2-c interactions with BAF and BAF-DNA complexes.
• To understand how DNA binding affects BAF conformation and its interaction with LAP2.

Main Methods:

• Site-directed mutagenesis of 17 Xenopus LAP2-c mutants.
• Assays for nuclear assembly inhibition in Xenopus egg extracts.
• In vitro binding assays for LAP2-c interaction with BAF and BAF-DNA complexes.

Main Results:

• Mutations within the LEM domain abolished both nuclear assembly inhibition and BAF binding.
• Mutations outside the LEM domain impaired nuclear assembly inhibition without affecting BAF binding, suggesting an additional function.
• LAP2 binding affinity was higher for BAF-DNA complexes than for BAF alone, and all isoforms supershifted BAF-DNA complexes.
• Mutagenesis indicated that BAF undergoes conformational changes upon DNA binding.

Conclusions:

• The LEM domain is essential for LAP2 binding to BAF.
• LAP2-c possesses functions beyond BAF interaction critical for nuclear assembly inhibition.
• Variable regions of LAP2 isoforms modulate the interaction with BAF, influencing binding affinities.
• LAP2 interactions with BAF-DNA complexes are physiologically significant for nuclear envelope regulation.