GTP Hydrolysis by Ran Is Required for Nuclear Envelope Assembly Martin Hetzer, Daniel Bilbao-Cortés, Tobias C Walther, Oliver J Gruss, Iain W Mattaj  Molecular Cell  Volume 5, Issue 6, Pages 1013-1024 (June 2000) DOI: 10.1016/S1097-2765(00)80266-X

Figure 1 A Two-Color Membrane Fusion Assay (A) Demembranated condensed sperm heads (top left) were incubated with purified nucleoplasmin to decondense the chromatin (top right). Subsequently, DiIC18 prelabeled membrane vesicles (red) were allowed to bind to chromatin, and the resulting sperm/membrane substrates were purified by centrifugation through a 30% sucrose cushion (bottom left). Upon addition of Xenopus egg cytosol, a closed nuclear envelope (CNE) formed that was visible as a rim around the completely decondensed chromatin in a confocal section (bottom right). DNA was stained with Hoechst 33342 dye (blue). (B) Membranes were separately labeled with DiIC18 (red-emitting) or DiOC18 (green-emitting) and purified with chromatin as described above either before (top two rows) or after (bottom row) mixing the two membrane preparations. Subsequently, the substrates were incubated with cytosol to allow nuclear formation. DiIC18 was visible only in the red channel and DiOC18 only in the green channel (top six panels). Mixing of the two differently labeled membrane vesicle populations gave rise to fusion of both types of labeled membrane and yellow NE staining in the overlay. Molecular Cell 2000 5, 1013-1024DOI: (10.1016/S1097-2765(00)80266-X)

Figure 2 The Ran Mutants, T24N and Q69L, Block Membrane Fusion on Chromatin (A) Decondensed sperm chromatin was incubated with DiIC18 (red) or DiOC18 (green)-labeled membrane vesicle preparations (see text). Purified chromatin-membrane substrates were incubated with cytosol in the presence of 2 mM GTPγS (top panels), 20 μM RanT24N (middle panels), or 20 μM RanQ69L (bottom panels). In the overlay, the red and green membrane populations remained essentially separated for up to 4 hr, indicating that the dyes did not diffuse significantly. The confocal sections show the upper surface of the chromatin. Nonfused vesicles are visible in large clusters. (B) After nuclear assembly, 60 confocal sections were taken along the z axis of the nucleus, deconvolved, and a three-dimensional image was calculated. A segment of a CNE (closed nuclear envelope) is shown from the nuclear exterior (left). The right panel shows nonfused vesicles covering an elongated stretch of chromatin (not visible) in the presence of GTPγS. Molecular Cell 2000 5, 1013-1024DOI: (10.1016/S1097-2765(00)80266-X)

Figure 5 Nuclear Assembly with λ DNA Substrates (A) λ DNA was incubated with fractionated Xenopus egg cytosol for 60 min at 20°C. Membranes, prelabeled with DiOC18, were then added for 90 min. Nuclear accumulation of rhodamine-labeled BSA-NLS or BSA-SLN conjugates was analyzed as indicated. Scale bar, 10 μm. (B) Left panel, λ DNA was assembled into chromatin in various extracts for 60 min. Membranes prelabeled with DiIC18 were then added and incubation continued for 90 min. Formation of CNEs occurred efficiently in mock depleted extract or in RCC1-depleted extract complemented with either RCC1 or Ran loaded with GTP. Ran loaded with GDP or GTPγS could not efficiently overcome the effect of RCC1 depletion. The numbers on the right represent the average percentage of CNEs formed on 50 randomly chosen chromatin templates in the various extracts in each of three independent experiments. Scale bar, 10 μm. Right panel, after chromatin assembly for 60 min, the assembly reaction was cooled briefly on ice, and labeled membranes were added and allowed to associate with the chromatin before rewarming. Equatorial sections are shown in all cases. Molecular Cell 2000 5, 1013-1024DOI: (10.1016/S1097-2765(00)80266-X)

Figure 3 Depletion of RCC1 Prevents the Formation of a CNE on Sperm Chromatin (A) One microliter of fractionated Xenopus egg extract (XTS), and either mock- or RCC1-depleted extract, as indicated, was analyzed by Western blotting with anti-RCC1 or anti-Ran antibodies. (B) Two-color chromatin-membrane substrates were incubated in mock- or RCC1-depleted extracts. The yellow NE formed in the mock extracts (top panels) is indicative of membrane fusion. CNE formation was inhibited in depleted extracts (middle panels) but could be restored by adding 400 nM RCC1 (bottom panels). Note that while CNE formation was blocked in the depleted extract, the two vesicle populations did not remain separated. (C) The formation of a CNE was scored for 60 randomly chosen chromatin substrates. In the mock-depleted extracts, almost 100% of the substrates were assembled into a CNE and subsequent nuclear growth occurred. In RCC1-depleted extracts, only 25% of substrates formed a CNE, but further nuclear growth was inhibited. The data for these columns is from four independent experiments. The presence of 2 μM RanT24N in the RCC1-depleted extracts blocked the residual activity and prevented fusion of red and green vesicles (two experiments). In contrast, the addition of either RanGDP or RanGTP (1 μM) to the RCC1-depleted extract significantly restored nuclear assembly (two experiments each). Molecular Cell 2000 5, 1013-1024DOI: (10.1016/S1097-2765(00)80266-X)

Figure 4 Active RCC1 Is Located on Sperm Chromatin (A) Western blot assay for the presence of RCC1 on demembranated sperm chromatin. Fractionated Xenopus egg cytosol (0.3 μl or 0.15 μl) (lanes 1 and 2) or chromatin from 2 × 105 or 1 × 105 sperm (lanes 3 and 4) was separated on an SDS gel and analyzed by Western blotting with anti-RCC1 antibody. (B) Assay of RCC1 exchange activity on sperm chromatin. [α-32P] GTP-loaded Ran was incubated with 2 mM GTP and the indicated concentrations of recombinant RCC1 or different amounts of decondensed sperm chromatin. (C) The concentration of RCC1 in high-speed Xenopus egg supernatant is roughly 200 nM (e.g.Dasso et al. 1994). The concentration of RCC1 on sperm chromatin was estimated from Western blots (A) and RCC1 exchange assays (B). The concentration was calculated on the basis of the volume of the decondensed sperm chromatin used in the assays. (D) Association of RCC1 with λ DNA-containing chromatin. λ DNA was added to mock-depleted Xenopus egg cytosol and assembled into chromatin. The chromatin was reisolated over a sucrose cushion, and proteins were extracted and separated by SDS-PAGE followed by Western blot analysis with anti-RCC1 or anti-U1A antibodies as indicated. Lane 1, no λ DNA; lane 2, 100 ng; lane 3, 200 ng; and lane 4, 400 ng λ DNA was added. Molecular Cell 2000 5, 1013-1024DOI: (10.1016/S1097-2765(00)80266-X)

Figure 6 Depletion of Ran Blocks NE Membrane Fusion (A) One microliter of mock- and Ran-depleted extracts was analyzed by Western blotting with anti-Ran or anti-importin-β antibody. (B) Depletion of Ran from NE assembly extracts blocked vesicle fusion on sperm chromatin (compare top and middle panels). Adding 2 μM RanGDP partially restored fusion (bottom panels). CNE formation was always significantly increased by addition of either RanGDP or RanGTP, but the efficiency of restoration varied depending on the depleted extract used (see text). (C) NE assembly was assayed using DiIC18 in mock- and Ran-depleted extract. DNA was counterstained with Hoechst dye. The ability of 2 μM Ran loaded either with GDP or GTPγS to restore CNE formation was assayed. Top panel, quantitation of CNE formation as a fraction of chromatin substrates. One hundred and fifty randomly chosen chromatin substrates were counted in each case. Bottom panel, representative images. (D) Nuclei were assembled in mock- or Ran-depleted extract or in Ran-depleted extract to which Ran loaded with GDP or GTP (2 μM), as indicated, had been added. DNA was visualized with Hoechst dye (blue) and nucleoporins with mAb414 (green) (Davis and Blobel 1987). Molecular Cell 2000 5, 1013-1024DOI: (10.1016/S1097-2765(00)80266-X)