Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Nuclear pore complex formation is a complex process involving membrane fusion and protein assembly.
• Annulate lamellae (AL), cytoplasmic organelles with pore complexes, can form spontaneously in Xenopus egg extracts.
• Understanding the mechanism of AL and nuclear pore assembly is crucial for cell biology research.

Purpose of the Study:

• To develop a biochemical assay for studying annulate lamellae formation.
• To investigate the mechanism of AL assembly and individual nucleoporin incorporation into pore complexes.
• To identify factors influencing nuclear pore protein association with membranes.

Main Methods:

• Developed a biochemical assay using Xenopus egg cytosol and membrane vesicles.
• Utilized immunoblotting to measure nucleoporin association with membranes.
• Employed thin-section electron microscopy, negative staining, and membrane flotation assays.
• Investigated the role of GTPγS and N-acetylglucosaminylated proteins in AL assembly.

Main Results:

• Specific nucleoporins (nup58, nup60, nup97, nup153, nup200) associated with membranes in a time- and temperature-dependent manner.
• Annulate lamellae formation was confirmed by electron microscopy and correlated with nucleoporin incorporation.
• A dense membrane fraction enriched in pore proteins (gp210 and nucleoporins) was isolated.
• GTPγS inhibited soluble pore protein incorporation; WGA-sepharose depletion altered AL structure and membrane density.

Conclusions:

• The developed biochemical assay effectively monitors annulate lamellae formation and nuclear pore assembly.
• Nuclear pore proteins incorporate into membranes during AL assembly, forming distinct dense membrane fractions.
• Specific factors, including N-acetylglucosaminylation, influence annulate lamellae structure and composition.