NUCLEAR-PORE COMPLEX ASSEMBLY STUDIED WITH A BIOCHEMICAL ASSAY FOR ANNULATE LAMELLAE FORMATION

Formation of the nuclear pore is an intricate process involving membra ne fusion and the ordered assembly of up to 1,000 pore proteins. As su ch, the study of pore assembly is not: a simple one. Interestingly, an nulate lamellae, a cytoplasmic organelle consisting of stacks of flatt ened membrane cisternae perforated by numerous pore complexes, have be en found to form spontaneously in a reconstitution system derived from Xenopus egg extracts, as determined by electron microscopy (Dabauvall e et al., 1991). In this work, a biochemical assay for annulate lamell ae (AL) formation was developed and used to study the mechanism of AL assembly in general and the assembly of individual nucleoporins into p ore complexes in particular. Upon incubation of Xenopus egg cytosol an d membrane vesicles, the nucleoporins nup58, nup60, nup97, nup153, and nup200 initially present in a disassembled form in the cytosol became associated with membranes and were pelletable. The association was ti me and temperature dependent and could be measured by immunoblotting. Thin-section electron microscopy as well as negative staining confirme d that annulate lamellae were forming coincident with the incorporatio n of pore proteins into membranes. Homogenization and subsequent flota tion of the membrane fraction allowed us to separate a population of d ense membranes, containing the integral membrane pore protein gp210 an d all other nucleoporins tested, from the bulk of cellular membranes. Electron microscopy indicated that annulate lamellae were enriched in this dense, pore protein-containing fraction. GTP gamma S prevented in corporation of the soluble pore proteins into membranes. To address wh ether AL form in the absence of N-acetylglucosaminylated pore proteins , AL assembly was carried out in WGA-sepharose-depleted cytosol. Under these conditions, annulate lamellae formed but were altered in appear ance. When the membrane fraction containing this altered AL was homoge nized and subjected to flotation, the pore protein-containing membrane s still sedimented in a distinct peak but were less dense than control annulate lamellae.