The balance of RanBP1 and RCC1 is critical for nuclear assembly and nuclear transport

Abstract

Ran is a small GTPase that is essential for nuclear transport, mRNA processing, maintenance of structural integrity of nuclei, and cell cycle control. RanBP1 is a highly conserved Ran guanine nucleotide dissociation inhibitor. We sought to use Xenopus egg extracts for the development of an in vitro assay for RanBP1 activity in nuclear assembly, protein import, and DNA replication. Surprisingly, when we used anti-RanBP1 antibodies to immunodeplete RanBP1 from Xenopus egg extracts, we found that the extracts were also depleted of RCC1, Ran's guanine nucleotide exchange factor, suggesting that these proteins form a stable complex. In contrast to previous observations using extracts that had been depleted of RCC1 only, extracts lacking both RanBP1 and RCC1 (codepleted extracts) did not exhibit defects in assays of nuclear assembly, nuclear transport, or DNA replication. Addition of either recombinant RanBP1 or RCC1 to codepleted extracts to restore only one of the depleted proteins caused abnormal nuclear assembly and inhibited nuclear transport and DNA replication in a manner that could be rescued be further addition of RCC1 or RanBP1, respectively. Exogenous mutant Ran proteins could partially rescue nuclear function in extracts without RanBP1 or without RCC1, in a manner that was correlated with their nucleotide binding state. These results suggest that little RanBP1 or RCC1 is required for nuclear assembly, nuclear import, or DNA replication in the absence of the other protein. The results further suggest that the balance of GTP- and GDP-Ran is critical for proper nuclear assembly and function in vitro.

Figure 2

Xenopus RanBP1 is a soluble Ran-binding protein. (A) RanBP1 is found in the cytosolic fraction of reconstituted extracts. Amounts of sperm chromatin, membranes, and cytosol equivalent to those in 1 μl of reconstituted extract were separated on SDS-PAGE followed by blotting to a PVDF membrane filter (lane 1, 1000 sperm nuclei; lane 2, 0.1 μl of membranes; lane 3, 0.9 μl of cytosol). Lane 4 contains 1.0 μl of the membrane fraction. The filter was stained with India ink (left lanes) followed by Western blotting with anti-Xenopus RanBP1 antibodies (right lanes). M represents protein molecular size standards in kilodaltons. (B) Xenopus RanBP1 binds GST-RanG19V. Egg cytosol (lanes 1 and 2), extracts of bacteria expressing Xenopus RanBP1 (lanes 3 and 4), or both (lane 5 and 6) were incubated with GST-RanG19V (lanes 1, 3, and 5) or GST (lanes 2, 4, and 6). Glutathione-Sepharose was added to the incubations to remove the GST-RanG19V- and GST-associated proteins. Proteins bound to the beads were eluted with sample buffer and separated by SDS-PAGE. Proteins from the gel were transferred to a PVDF membrane, and Western blotting analysis was performed with anti-human RanBP1 antibodies. (Identical results were obtained when the experiment was performed with anti-Xenopus RanBP1 antibodies; our unpublished results). Lane 7 contained 1 μl of egg cytosol. GST-RanG19V was preloaded with GTP prior to this experiment. Both wild-type and his-tagged RanBP1 proteins also bound to wild-type GST-Ran but with a lower affinity that probably reflects the fact that wild-type Ran would exist as a mixed population of GTP- and GDP-bound forms (our unpublished results).

Figure 7

Partial rescue of nuclear assembly and function in RCC1 or RanBP1 deficient extracts by Ran mutants. (A) Nuclear protein import can be rescued by Ran mutants. XB buffer (second row from top), RanBP1 (50 μg/ml; third row), or RCC1 (10 μg/ml; bottom row) was added to codepleted extracts in the absence (two left columns) or presence of RanG19V (middle two columns; 40 μg/ml) or RanT24N (right two columns; 40 μg/ml) proteins. Similar samples were prepared with mock-depleted extracts (top row). The extracts were incubated on ice for 15 min, and standard nuclear assembly assays were performed. Rhodamine-labeled protein import substrate were added after 60 min. The samples were examined 120 min after the start of the assembly reaction. The parts with uppercase locants show typical nuclei from each reaction obtained with Hoechst 33258 DNA dye and with the lowercase locants show the accumulation of nuclear transport substrate in the same nuclei. Bar, 3.0 × 10⁻⁶ m. (B) DNA replication was partially restored by Ran mutants. In the same nuclear assembly assay as in A, samples were taken after 120 min for DNA replication assays and results were quantified on a Phosphorimager.

Figure 1

DNA and protein sequences of Xenopus RanBP1. (A) DNA sequence of Xenopus RanBP1 cDNA. Lowercase type at both ends of the nucleotide sequence shows the restriction sites for EcoRI (5′) and XhoI (3′). The numbers correspond to nucleotide sequences (normal type) and amino acid sequences (boldface type). The underlined nucleotides indicate stop codons preceding the putative initiation codon. (B) Alignment of RanBP1 homologues. The protein encoded by the open reading frame for the Xenopus RanBP1 sequence (XenBP1, GenBank accession number AF015303) was aligned with RanBP1 proteins of human (HumBP1, GenBank accession number X83617), mouse (MusBP1, accession number L25255), and S. cerevisiae (Yrb1p, accession number X65925). Amino acids that are identical among the four homologues are indicated by type in the consensus sequence (CONSEN). The star indicates the amino acids that are identical among human, mouse, and Xenopus RanBP1 and the tilde indicates the other amino acids that are identical in any three homologues. Gaps inserted for optimal sequence alignment are indicated by periods.

Figure 3

Depletion of RanBP1 results in the codepletion of RCC1. (A) Anti-RanBP1 antibodies immunoprecipitate both RanBP1 and RCC1. Immunoprecipitates of anti-RanBP1 (lane 2) or preimmune sera (lane 1) were analyzed by SDS-PAGE together with 1 μl egg extract (lane 3). M indicates molecular weight marker. Top, a silver stained gel of the samples, with the position of RCC1 and RanBP1 indicated. Bottom, duplicate samples analyzed by Western blotting with anti-RCC1 and anti-RanBP1 antibodies. The faint band in lane 1 of the anti-RCC1 Western blot is the immunoglobulin heavy chain. (B) RCC1 is specifically and quantitatively codepleted with RanBP1. One μl of control (lane 1), immunodepleted (lane 2), and mock depleted (lane 3) cytosol were subjected to SDS-PAGE and Western blotting analysis with antibodies against importin β, RanGAP1, RCC1, RanBP1, histone B4, and Ran as indicated. (C) No RanBP1-like proteins remain in Xenopus egg cytosol after RanBP1 immunodepletion. One-microliter samples of control (lane 1), immunodepleted (lane 2), and mock-depleted extracts were subjected to SDS-PAGE and transferred to a PVDF membrane. The filter was incubated with [α-³²P]GTP-bound GST-Ran to allow RanBP1 detection (see MATERIALS AND METHODS). RanBP1-depleted extracts show no low molecular weight Ran-binding proteins in this assay.

Figure 4

Normal nuclear assembly and function in the absence of RanBP1 and RCC1. (A) Nuclear assembly occurs normally in the absence of both RanBP1 and RCC1. Control (top), codepleted (middle), and mock-depleted (bottom) cytosol were used for a standard nuclear assembly assays. After 30, 60, and 120 min of incubation, nuclei were stained with Hoechst 33258 DNA dye. Bar, 2.6 × 10⁻⁶ m. (B) DNA replication occurs normally in the absence of both RanBP1 and RCC1. Nuclei assembled as in A were allowed to carry out DNA replication in the presence of [α-³²P]dCTP. At 90 min and 150 min, DNA samples from control (row 1), codepleted (row 2), and mock-depleted (row 3) reactions were taken for analysis of ³²P incorporation on agarose gels. The samples were treated as previously described (Smythe and Newport, 1991), and replication was quantified using a Molecular Dynamics Phosphorimager.

Figure 6

Balance of RCC1 and RanBP1 is critical for nuclear assembly and function. (A) Imbalance of RanBP1 and RCC1 causes defects in nuclear assembly and protein import. Equal volumes of XB Buffer (row 1), recombinant RCC1 (final concentration = 10 μg/ml; row 2), recombinant RanBP1 (final concentration = 50–70 μg/ml; row 3), or both RCC1 and RanBP1 (row 4) were added to codepleted cytosol and incubated for 15 min on ice. The cytosol was then used in nuclear assembly reactions. A rhodamine-labeled import substrate was added 60 min after nuclear assembly began, and 120 min after the reaction began, each sample was assayed for nuclear morphology and protein import. Phase-contrast images of typical nuclei are shown on the left; corresponding photographs of nuclear DNA (stained with Hoechst 33258) and protein import assays are shown in the middle and right, respectively. Bar, 2.6 × 10⁻⁶ m. [As a control, BSA was added to codepleted extracts. BSA neither inhibited nuclear assembly in codepleted extracts nor restored nuclear assembly in codepleted extracts to which either RanBP1 or RCC1 had been added (our unpublished results).] (B) Quantitation of protein import and nuclear size among control and experimental samples. A set of samples similar to those in A (bars 1–4) plus a control (bar C) was allowed to form nuclei under the conditions described above. After 90 min, images of at least 25 nuclei from each sample were randomly selected and captured by using an IP Labs Spectrum Imaging System with a Photometrics cooled charge-coupled device camera. Capture was performed under identical conditions for each sample and the intensity of the signal was not saturating the imaging system. Nuclear size was measured as the number of pixels occupied by the nucleus at its maximal cross-sectional area. Import was measured as pixel intensity within the nucleus, corrected for background fluorescence. The mean and standard deviations were calculated to obtain the relative levels of import (top) and nuclear size (bottom). (C) RanBP1 and RCC1 protein levels. A 1-μl volume from each of the nuclear assembly reactions shown in A was subjected to SDS-PAGE and Western blot analysis with anti-RCC1 or anti-Xenopus RanBP1 antibodies, as indicated. Lane C shows the amount and positions of endogenous RCC1, RanBP1, and added recombinant RanBP1 (bigger than endogenous RanBP1 because of the tag). (D) DNA replication is inhibited by the addition of either RCC1 or RanBP1 but restored by the addition of both. Reactions of a control extract and those shown in A were allowed to undergo DNA replication in the presence of [α-³²P]dCTP. At 180 min, samples from the control (bar C) and reactions of codepleted extract containing XB buffer (bar 1), recombinant RCC1 (bar 2), recombinant RanBP1 (bar 3), or both RCC1 and RanBP1 (bar 4) were taken for analysis of ³²P incorporation. The samples were treated as previously described (Smythe and Newport, 1991) and the amount of ³²P incorporated into high molecular weight DNA was quantified using a Phosphorimager.

Figure 5

Cyclin B-induced mitotic events occur normally in the absence of RanBP1 and RCC1. (A) The activation of histone H1 kinase is not affected by the depletion of RanBP1 and RCC1. Nuclear assembly reactions were reconstituted with untreated (row 1), codepleted (row 2), or mock-depleted (row 3) cytosol. Nuclear formation was allowed to proceed for 60 min before addition of nondegradable cyclin B to initiate mitosis in half of the reaction mix (+ Cyclin B). The remainder of the sample was allowed to continue in the absence of cyclin B (− Cyclin B). At the times after cyclin B addition indicated (minutes), samples from each reaction were assayed for the H1 kinase activity. We observed that histone H1 kinase activity was induced synchronously in all of the reactions containing cyclin B. (B) The induction of NEBD and chromosome condensation are not affected by the depletion of RanBP1 and RCC1. Nuclear assembly was allowed to proceed for 60 min in extracts reconstituted with untreated (top), codepleted (middle), or mock-depleted (bottom) cytosol. Nondegradable cyclin B was added to half (left) of each nuclear assembly reaction, and buffer was added to the other half of the reaction (right). Nuclear breakdown occurred synchronously in the samples with cyclin B, and the nuclei remained intact in the samples without cyclin B. Each sample was stained with Hoechst 33258 DNA dye 90 min after cyclin B addition and photographed by fluorescence microscopy. Bar, 2.6 × 10 ⁻⁶ m.