Purification of the Vertebrate Nuclear Pore Complex by Biochemical Criteria

Abstract

The nuclear pore is a large and complex biological machine, mediating all signal-directed transport between the nucleus and the cytoplasm. The vertebrate pore has a mass of ~120 million daltons or 30 times the size of a ribosome. The large size of the pore, coupled to its tight integration in the nuclear lamina, has hampered the isolation of pore complexes from vertebrate sources. We have now developed a strategy for the purification of nuclear pores from in vitro assembled annulate lamellae (AL), a cytoplasmic mimic of the nuclear envelope that lacks a lamina, nuclear matrix, and chromatin-associated proteins. We find that purified pore complexes from annulate lamellae contain every nuclear pore protein tested. In addition, immunoblotting reveals the presence of soluble transport receptors and factors known to play important roles in the transport of macromolecules through the pore. While transport factors such as Ran and NTF2 show only transient interaction with the pores, a number of soluble transport receptors, including importin β, show a tight association with the purified pores. In summary, we report that we have purified the vertebrate pore by biochemical criteria; silver staining reveals ~40-50 distinct protein bands. KeywordsNuclear pore; annulate lamellae; purification; nucleoporin; importin; vertebrate The regulated transport of macromolecules across the nuclear envelope is critical for the creation and maintenance of the discrete identities of the nucleus and cytosol. Traffic through the nuclear envelope utilizes the nuclear pore complex, a large proteinaceous structure in excess of 120 million daltons in mass (1-3). As such, the nuclear pore represents one of the largest biological machines within the cell. The pore itself consists of three stacked octagonally symmetric rings, with the central ring composed of spoke-like structures supporting a central transporter ( Figure 1B). Extending from the cytoplasmic ring are eight short filaments (1,2,4,5). These filaments, composed in part of the nuclear pore proteins (or nucleoporins) Nup214/CAN and Nup358/RanBP2, contain the initial binding sights for nuclear import receptors and their cargo (6)(7)(8)(9)(10)(11)(12)(13)(14). Extending from the nuclear ring are eight thin filaments terminating in a smaller ring. This nuclear pore 'basket' minimally contains the nucleoporins Nup93, Nup98, and Nup153, and plays roles both in export from the

Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

“…As expected, the cytoplasmic pore complexes labeled by mAb414 were almost completely overlapped with the staining of RanBP2, which was used a marker for the RanBP2/RanGAP1*SUMO1/Ubc9 complexes ( Fig 4B ). Consistent with the previous studies [ 12 , 60 , 62 ], we found that ~98% of the mAb414-labeled cytoplasmic foci of ALPCs are associated with the network of ER stained by anti-calreticulin antibody after analyzing 50 cells (~12 foci/cell) ( Fig 4A ).…”

“…Several karyopherins, including importin α/β and CRM1, have been shown previously to be associated with both NPCs and ALPCs in Xenopus eggs and mouse embryonic fibroblasts [ 60 , 62 , 69 ]. We therefore further hypothesized that the partition of importin α/β and CRM1 between NPCs and ALPCs is also determined by the relative levels of these two pore complexes.…”

Ankyrin-G induces nucleoporin Nup358 to associate with the axon initial segment of neurons

“…S1A). This was further confirmed by immunostaining neurons for specific nucleoporins known to be incorporated into AL such as Nup98 (Miller and Forbes, 2000) and more importantly, Nup88 (Wu et al, 2001) that interacts with Nup358 at the cytoplasmic side of the NPC (Bernad et al, 2004) (Fig. S1B,C).…”

Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export

“…We examined assembled pores for the Nup160 complex. Annulate lamellae (AL), cytoplasmic stacks of membranes that contain abundant pores identical to nuclear pores, can easily be assembled in Xenopus extracts in vitro ( Dabauvalle et al, 1991 ; Cordes et al, 1995 ; Meier et al, 1995 ; Miller et al, 2000 ; Miller and Forbes, 2000 ). AL were formed in egg extract, purified, and treated with 0.5 M NaCl to partially solubilize the pores ( Miller et al, 2000 ).…”