JCS039529 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A) Incubation of chromatin with cytosol and the combined heavy membranes (labeled with DilC₁₈, red) and light membranes (labeled with DiOC₁₈, green) results in nuclei with a pore-containing NE. Fluorescence microscopy reveals recruitment of both membrane types to the nuclear periphery (upper row). The presence of NPCs is evident by anti-Nup62 staining and nuclear transport of fibrillarin (bottom row). Bars, 10 µm.

  (B) Electron micrograph showing part of a nucleus which was reconstituted as described in (A). A double nuclear membrane surrounds the electron-dense chromatin, either in direct contact or at some distance. The cisternal space is perforated at multiple sites by NPCs (arrows). Bar, 0.1 µm.

• Supplemental Figure S2 -

  Fig. S2. Immoblot analysis of the light and heavy membranes fraction using antibodies against the transmembrane nucleoporins gp210 and POM121, the nuclear import receptors importin α and β, the small GTPase Ran, the lamin B receptor (LBR), a transmembrane protein of the inner nuclear membrane, and p97ATPase which belongs to the AAA-familiy of ATPases and is involved in fusion processes of several membrane types including the nuclear membrane. The only difference found with this set of antibodies is the apparent absence of Ran from the light membranes.

• Supplemental Figure S3 -

  Fig. S3. MVP/vaults are attached to the light membranes as shown by flotation centrifugation. An aliquot of the light membranes was adjusted to 60% sucrose and overlaid with 40% and 30% sucrose as described in Materials and Methods. After centrifugation, the three fractions with different sucrose concentrations were collected from the top and probed with antibodies to MVP. Antibodies against the integral nucleoporin POM121 and LBR, a transmembrane protein of the inner nuclear membrane, served as markers. The majority of MVP floated together with membranes into the overlying 40% sucrose layer. Interestingly, membranes recovered from the 30% sucrose layer contained POM121 and LBR, but lacked MVP. Possibly due to the relatively short centrifugation times, some MVP remained in the 70% sucrose layer, most likely in association with LBR-containing membranes.

• Supplemental Figure S4 -

  Fig. S4. A large fraction of endogenous vaults present in the crude egg extract is associated with membranes. (A) Scheme depicting the preparation of crude extract from Xenopus eggs. Centrifugation of the crude extract at 100,000 g for 1 hr yielded the S100 supernatant which was used for the isolation of membranes as indicated in text Fig. 1A and a pellet (P100). Although free vault particles sediment under these conditions, only a minor fraction was recovered in P100 as shown by the Western blot (B). Vaults remaining in the supernatant fraction (S100) were apparently membrane bound. When the membranes were solubilized by adding 2% Triton X-100 to the crude extract followed by centrifugation, the majority of vaults were now recovered in the pellet (B).

• Supplemental Figure S5 -

  Fig. S5. The NE of control nuclei and AL are stained with antibodies to MVP, but not the pore-free nuclei. Chromatin was incubated either in control extract (upper panel) or in preincubated extract to allow formation of AL lower panel; for details see Ewald et al. (Ewald et al., 1997). Incubation in control extract results in the assembly of nuclei surrounded by an intact NE with NPCs (i.e. Nup62 positive). These control nuclei score positive for MVP (upper row). In contrast, preincubated extract promotes the assembly of NPC-free nuclei (Nup62 negative; lower row). Poreless nuclei are also negative for MVP (one nucleus is easily identified by phase contrast and Hoechst staining). Numerous distinct cytoplasmic aggregates are visible in the centrifuged preparation which strongly fluoresce with antibodies to Nup62 and hence most likely represent AL stacks (see Dabauvalle et al., 1991). Interestingly, the AL score positive for MVP (lower row). Bar, 10 µm.

• Supplemental Figure S6 -

  Fig. S6. Incubation of recombinant xMVP with cytosol. After 10 min of incubation the sample was centrifuged at 100,000 g for 1 hr and the resulting supernatant (S100) and pellet (P100) were probed with antibodies to MVP. A large fraction of MVP is recovered in the pellet fraction indicating the assembly of vault particles. Both MVP-containing fractions promoted the insertion of NPC into pore-free nuclei.