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. 2006 May;26(10):3798-809.
doi: 10.1128/MCB.26.10.3798-3809.2006.

Nucleophosmin is essential for ribosomal protein L5 nuclear export

Yue Yu  1 , Leonard B Maggi Jr, Suzanne N Brady, Anthony J Apicelli, Mu-Shui Dai, Hua Lu, Jason D Weber
Affiliations

Nucleophosmin is essential for ribosomal protein L5 nuclear export

Yue Yu et al. Mol Cell Biol. 2006 May.

Abstract

Nucleophosmin (NPM/B23) is a key regulator in the regulation of a number of processes including centrosome duplication, maintenance of genomic integrity, and ribosome biogenesis. While the mechanisms underlying NPM function are largely uncharacterized, NPM loss results in severe dysregulation of developmental and growth-related events. We show that NPM utilizes a conserved CRM1-dependent nuclear export sequence in its amino terminus to enable its shuttling between the nucleolus/nucleus and cytoplasm. In search of NPM trafficking targets, we biochemically purified NPM-bound protein complexes from HeLa cell lysates. Consistent with NPM's proposed role in ribosome biogenesis, we isolated ribosomal protein L5 (rpL5), a known chaperone for the 5S rRNA. Direct interaction of NPM with rpL5 mediated the colocalization of NPM with maturing nuclear 60S ribosomal subunits, as well as newly exported and assembled 80S ribosomes and polysomes. Inhibition of NPM shuttling or loss of NPM blocked the nuclear export of rpL5 and 5S rRNA, resulting in cell cycle arrest and demonstrating that NPM and its nuclear export provide a unique and necessary chaperoning activity to rpL5/5S.

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Figures

FIG. 1.
FIG. 1.
Nuclear export of NPM is CRM1 dependent. NIH 3T3 cells were seeded onto HeLa cells that had been transfected with His-NPM in combination with Myc-NPC-M9 (shuttling control) in the (A) absence or (B) presence of LMB. Heterokaryons were incubated in media containing cycloheximide for an additional 4 h before fixation. Heterokaryon formation was verified by phase-contrast microscopy, while His-NPM and Myc-NPC-M9 proteins were visualized with antibodies against His (red) and Myc (green), respectively. DNA was stained with Hoechst. Mouse nuclei are demarcated with dotted circles. Human and mouse nuclei are labeled h and m, respectively. These data are representative of at least five independent heterokaryons formed for each transfection condition in three independent experiments. The percentages of His-NPM shuttling in heterokaryons are given. DIC, differential interference contrast; α, anti. (C) Sequence alignment of putative NPM NESs with known NESs of CRM1-dependent nuclear export proteins (p53, protein kinase inhibitor [PKI], rev, and Mdm2). Critical hydrophobic residues are indicated in yellow.
FIG. 2.
FIG. 2.
Leucine 42 and leucine 44 are identified as critical nuclear export residues. NIH 3T3 cells were seeded onto HeLa cells that had been transfected with (A) His-NPMΔ42-61, (B) His-NPMΔ62-83, or (C) NPMdL in combination with Myc-NPC-M9. Ectopic NPM proteins and Myc-NPC-M9 proteins were visualized with antibodies against His (red) and Myc (green), respectively. DNA was stained with Hoechst. Mouse nuclei are demarcated with dotted circles. Human and mouse nuclei are labeled h and m, respectively. These data are representative of at least five independent heterokaryons formed for each transfection condition in three independent experiments. The percentages of His-NPM shuttling in heterokaryons are given. DIC, differential interference contrast; α, anti. (D) Sequence alignment of NPM homologues throughout evolution. Identical residues in all species are marked yellow, identical residues in at least seven species are highlighted blue, and conserved residues are marked green. Crystal structure features are identified above the sequences. The consensus NPM sequence for all 11 identified homologues is given, with conserved nuclear export leucines 42 and 44 marked with arrows (NES).
FIG. 3.
FIG. 3.
NPM shuttling mutants act as dominant negative inhibitors of NPM nuclear export. (A, left) HeLa cells transduced with His-NPMdL and GFP-NPM were lysed, and the whole-cell lysate was subjected to immunoprecipitation (IP) with NRS or antibodies recognizing His and GFP epitopes. Precipitated protein complexes were separated by SDS-PAGE, and ectopic NPM proteins were visualized with antibodies against GFP and His epitopes. (A, right) HeLa cells transfected with His-NPMdL were lysed, and the whole-cell lysate was subjected to immunoprecipitation with NRS or antibodies recognizing His epitopes. Precipitated protein complexes were separated by SDS-PAGE, and ectopic mutant and endogenous wild-type NPM proteins were visualized with antibodies against NPM. Untransfected HeLa whole-cell lysate was loaded as a marker for endogenous NPM expression (lane 1). IB, immunoblot; α, anti; DIC, differential interference contrast. NIH 3T3 cells were seeded onto HeLa cells that had been transfected with GFP-NPM (B) alone or (C) in combination with His-NPMdL. Heterokaryon assays were performed, and His-NPMdL and GFP-NPM proteins were visualized with antibodies against His (red) or naturally emitting GFP spectra (green). DNA was stained with Hoechst. Mouse nuclei are demarcated with dotted circles. Human and mouse nuclei are labeled h and m, respectively. These data are representative of at least five independent heterokaryons formed for each transfection condition in three independent experiments. The percentages of GFP-NPM shuttling in heterokaryons are given.
FIG. 4.
FIG. 4.
Isolation of endogenous NPM protein complexes. (A) HeLa cell lysates (600 μg) transduced with control (−) or NPM-directed RNA interference (RNAi) constructs (+) were injected onto either an NRS column or custom NPM polyclonal antibody affinity columns and eluted with an increasing NaCl gradient (0.1 to 1.0 M). Eluted proteins were separated by SDS-PAGE and visualized with Coomassie blue stain. Identified bands are labeled. (B) Representative MALDI-TOF spectra of labeled protein bands from panel A are shown with labeled matching peptide masses.
FIG. 5.
FIG. 5.
NPM interacts directly with rpL5. (A, left) Recombinant NPM, NPMdL, and rpL5 were purified from bacterial lysates using Ni-nitrilotriacetic acid affinity chromatography. Purified proteins were separated by SDS-PAGE and detected with Coomassie blue stain. (A, middle) Purified NPM or NPMdL proteins were incubated overnight with rpL5 and immunoprecipitated (IP) with NRS or antibodies recognizing NPM or rpL5. Precipitated proteins were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with NPM and rpL5 antibodies. (A, left) Purified NPM or rpL5 proteins were incubated overnight with recombinant p27 and immunoprecipitated with NRS or antibodies recognizing NPM, rpL5, or p27. Precipitated proteins were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with NPM, rpL5, and p27 antibodies. α, anti. (B) Proteins from HeLa cell lysates were immunoprecipitated with NRS, rpL5 antibody, or NPM antibodies. Precipitated proteins were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with NPM and rpL5 antibodies. Alternatively, HeLa lysates were pretreated for 1 h with RNase A prior to immunoprecipitation as described above. (C) HeLa lysates were subjected to serial immunoprecipitation with NPM antibodies (lanes 1o to 4o). Precipitated proteins and proteins in the final supernatant (unbound) were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with antibodies recognizing NPM and rpL5.
FIG. 6.
FIG. 6.
NPM and rpL5 colocalize with nuclear and cytosolic ribosome subunits. HeLa cells transduced with vector (top panels) or His-NPMdL (middle panels) or treated with LMB (bottom panels) were divided into cytoplasmic and nuclear fractions and subjected to sucrose gradient centrifugation. Absorbance was monitored at 254 nm, and fractions containing 40S, 60S, 80S, and polysome units were collected. Proteins from each fraction were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with antibodies recognizing NPM, the His epitope, and rpL5. α, anti.
FIG. 7.
FIG. 7.
NPM nuclear export signals are required for the efficient export of GFP-rpL5. (A) HeLa cells either untransfected or transfected with GFP-tagged L5 for 48 h were harvested and lysed. Proteins were immunoprecipitated (IP) with NRS or a rabbit GFP antibody. Precipitated proteins were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with GFP and NPM antibodies. α, anti; DIC, differential interference contrast. Loading inputs are indicated. (B to E) NIH 3T3 cells were seeded onto HeLa cells that had been transfected with GFP-rpL5 in combination with (B and C) His-NPM, (D) His-NPMΔ42-61, and (E) His-NPMdL. Additionally, HeLa cells in panel C were treated with LMB for 18 h prior to fusion. Heterokaryon assays were performed with NPM and GFP-rpL5 proteins being visualized with antibodies against His (red) and naturally emitting GFP spectra (green), respectively. DNA was stained with Hoechst. Mouse nuclei are demarcated with dotted circles. Human and mouse nuclei are labeled h and m, respectively. These data are representative of at least five independent heterokaryons formed in three independent experiments. The percentages of heterokaryons exhibiting GFP-rpL5 shuttling are given.
FIG. 8.
FIG. 8.
NPM is essential for rpL5 nuclear export. (A) HeLa cells (−) or cells transduced with siRNAs encoding either scrambled control or NPM-specific sequences were harvested 72 h posttransduction for Western blot analysis. Proteins separated by SDS-PAGE were transferred to PVDF membranes and immunoblotted with antibodies recognizing NPM and γ-tubulin. α, anti. (B) HeLa cells (−) or cells transduced with siRNAs encoding either scrambled control or NPM-specific sequences were harvested 72 h posttransduction for cellular fractionation. Proteins from nuclear (N) and cytosolic (C) fractions were analyzed by SDS-PAGE and immunoblotted with antibodies recognizing rpL5, SOD (cytoplasm control), and lamin A/C (nuclear control). (C) HeLa cells were transfected with His-NPM or His-NPMdL, and 24 h later equal numbers of cells were subjected to fractionation into cytoplasmic (C) and nuclear (N) extracts. L5 protein was detected by Western blot analysis (top panels; WB). Lamin A/C and SOD are shown as nuclear and cytoplasmic fractionation controls, respectively (top panels; WB). 5S rRNA was detected by Northern blot analysis of total RNA extracted from the nuclear and cytosolic fractions (bottom panel; NB). The ratios of nuclear to cytoplasmic accumulation of rpL5 and 5S rRNA are given as percentages of totals (*, P > 0.001). (D) HeLa cells were transfected with vector, His-NPM, or His-NPMdL, and cells were plated on glass coverslips. Twenty-four hours later, cells were subjected to RNA FISH with a TRITC-labeled 5S rRNA probe. Nuclei were stained with DAPI. Untransfected HeLa cells were treated with LMB for 18 h prior to RNA FISH analysis. Results of 5S rRNA localization are each representative of three independent experiments. (E) Wild-type (WT) MEFs were infected with control retroviruses or those encoding ARF, and 48 h later equal numbers of cells were subjected to fractionation into cytoplasmic (C) and nuclear (N) extracts. 5S rRNA was detected by Northern blot analysis of total RNA extracted from the nuclear and cytosolic fractions (*, P > 0.005). (F) HeLa cells were transfected with vector, His-NPM, His-NPMdL, or His-NPMΔ42-61 and plated on glass coverslips. Cells were incubated with BrdU 72 h posttransfection and fixed 20 h later. Fixed cells were stained with antibodies recognizing BrdU and His epitopes and visualized by immunofluorescence using fluorescein isothiocyanate- and TRITC-labeled secondary antibodies, respectively. Cells (100) were counted for each condition in triplicate. Standard deviations are reported as error bars (*, P > 0.005).
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