Savulescu AF et al. (2011), Nuclear import of an intact preassembled protea...

XB-ART-42756

Mol Biol Cell 2011 Mar 15;226:880-91. doi: 10.1091/mbc.E10-07-0595.

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Nuclear import of an intact preassembled proteasome particle.

Savulescu AF , Shorer H , Kleifeld O , Cohen I , Gruber R , Glickman MH , Harel A .

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The 26S proteasome is a conserved 2.5 MDa protein degradation machine that localizes to different cellular compartments, including the nucleus. Little is known about the specific targeting mechanisms of proteasomes in eukaryotic cells. We used a cell-free nuclear reconstitution system to test for nuclear targeting and import of distinct proteasome species. Three types of stable, proteolytically active proteasomes particles were purified from Xenopus egg cytosol. Two of these, the 26S holoenzyme and the 20S core particle, were targeted to the nuclear periphery but did not reach the nucleoplasm. This targeting depends on the presence of mature nuclear pore complexes (NPCs) in the nuclear envelope. A third, novel form, designated here as 20S+, was actively imported through NPCs. The 20S+ proteasome particle resembles recently described structural intermediates from other systems. Nuclear import of this particle requires functional NPCs, but it is not directly regulated by the Ran GTPase cycle. The mere presence of the associated "+" factors is sufficient to reconstitute nuclear targeting and confer onto isolated 20S core particles the ability to be imported. Stable 20S+ particles found in unfertilized eggs may provide a means for quick mobilization of existing proteasome particles into newly formed nuclear compartments during early development.

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Species referenced: Xenopus laevis
Genes referenced: actl6a dnai1 hsp90aa1 ran ranbp2 rpn2

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	FIGURE 1:. Nuclear targeting of proteasomes. (A) In vitro reconstituted nuclei were separated from the surrounding cytosol by centrifugation through a sucrose cushion, as described in Materials and Methods. Total reaction, cytosolic, and nuclear fractions were immunoblotted with specific markers. Note that actin is exclusively cytosolic, while a fraction of the histone and nucleoporin markers are recruited to nuclei within the time of incubation. The Î±7 and Rpn2 proteasome subunits are also significantly recruited to the nuclear fraction. (B) Reconstituted nuclei were fixed and processed for indirect immunofluorescence. DNA was stained with Hoechst 33258 (left column). Anti-Nup358 exhibits the typical punctuate rim staining of NPC components. Both Î±7 and Rpn2 antibodies exhibit punctuate staining throughout the nucleus. Scale bar, 10 Î¼m.
	FIGURE 2:. Proteasome targeting requires mature nuclear envelopes and NPCs. (A) Schematic representation of major steps and known chemical inhibitors of the nuclear assembly pathway (reviewed by Harel and Forbes, 2004). The addition of GTPÎ³S at t = 0 of nuclear reconstitution results in unfused membrane vesicles docked on the surface of chromatin. BAPTA inhibits a subsequent step, resulting in aborted assembly intermediates with sealed double nuclear membranes, but no NPCs (poreless nuclei). If nuclear assembly is allowed to proceed normally and recombinant ImpÎ² 45â462 is later added, mature NPCs are formed, but nuclear transport is blocked (plugged pores). (B) Separation of nuclear and cytosolic fractions was carried out as in Figure 1A and compared between normal nuclei (left three columns) and BAPTA-inhibited intermediates (right three columns). Proteasome markers were missing in BAPTA-inhibited nuclear intermediates. (C) Normal (functional) nuclei and BAPTA-inhibited intermediates were immunostained with anti-Î±7 as in Figure 1B. Scale bar, 10 Î¼m. (D) Separated nuclear and cytosolic fractions were compared between normal assembly and GTPÎ³S-inhibited reactions. The Î±7 proteasomal marker is absent from the aborted assembly intermediates.
	FIGURE 3:. Biochemical purification of three distinct species of proteasome particles. (A) Migration of endogenous proteasomes (extract) and purified particles (designated 26S, 20S+, and 20S) in native gels. Active particles were visualized by the Suc-LLVY-AMC peptide, which becomes fluorescent upon proteolytic cleavage in the internal cavity of the proteasome core particle. The slower migrating top two bands correspond to single- and double-capped 26S forms known from yeast extracts and other systems. The 20S+ particle shows higher peptidase activity than an equivalent sample of the 20S CP. (B) Silver staining (top) and immunoblot analysis (bottom) of samples of the three purified particle preparations, following denaturing SDSâPAGE. The blot was probed with antibodies directed against Î±7 (a 20S CP subunit), Rpn2 and Rpt1 (19S subunits), and Hsp90, which is only detected in the 20S+ particle. (C) Identical samples of the three purified particle preparations (26S, 20S+, and 20S) were loaded for the in-gel peptidase activity assay shown in (A) and in three native gels used for immunoblotting. Equal loading based on anti-Î±7 reactivity in a denaturing immunoblot is shown in the bottom strip of (A). The native blots were probed against Î±7, Rpn2, and Hsp90, all of which were detected in the single band of the 20S+ particle. (D) Immunoprecipitation out of Xenopus egg cytosol was performed with affinity-purified antibodies generated against the central PC-repeat domain of xRpn2, or control rabbit IgG. Rpn2 and Î±7 were coimmunoprecipitated, but Rpt1 was missing, indicating the presence of an endogenous particle in the extract containing the 20S CP together with an exposed Rpn2 subunit.
	FIGURE 4:. Nuclear targeting and import of fluorescently labeled proteasomes. Purified 26S, 20S+, and 20S particles were fluorescently labeled and added into nuclear reconstitution reactions after the assembly of functional nuclei had been completed. Samples were fixed and analyzed by confocal microscopy. For each of the three particles a representative nucleus is shown, starting with a surface (top) view section on the left, and followed by three consecutive 1-Î¼m-thick confocal sections through the middle of the nucleus. The labeled 26S and 20S particles accumulate at the nuclear envelope, while the 20S+ particle reaches the nucleoplasm.
	FIGURE 5:. Nuclear targeting of the 20S+ particle requires functional NPCs. (A) Labeled 20S+ particles were added into normal reconstitution reactions (functional nuclei), or reactions in which NPC assembly was inhibited by BAPTA (poreless nuclei). Samples were fixed and analyzed by epifluorescence microscopy; 20S+ staining was not observed in BAPTA-inhibited intermediates. (B) Normal reconstituted reactions were split in two and 8 Î¼M ImpÎ² 45â462 was added to plug the channel of preassembled NPCs in one sample. Labeled 20S+ particles were then added to both samples. Consecutive confocal sections through the middle of representative nuclei are shown. The 20S+ accumulated inside normal nuclei, but only stained the nuclear envelope when pores were plugged. (C) Nuclear and cytosolic fractions were separated and immunoblotted with the Î±7 proteasomal marker, as in Figure 2. Normal nuclei were compared with BAPTA-inhibited nuclei [corresponding to poreless intermediates in (A)], and ImpÎ² 45â462 was added after assembly [plugged pores in (B)]. The Î±7 subunit is not detected in the nuclear fraction of BAPTA-inhibited intermediates and a smaller portion of Î±7 cofractionates with plugged-pore nuclei as compared with normal nuclei.
	FIGURE 6:. 20S+ import is not blocked by RanGTP. (A) Reconstituted nuclei were preincubated with buffer (control) or 10 Î¼M RanQ69L-GTP for 15 min, before the addition of fluorescently labeled 20S+ particles and TRITC-NLS-BSA import substrate. Both 20S+ and the NLS cargo accumulated in control nuclei (top row; confocal midsection). The import of the NLS cargo was blocked in the presence of RanQ69L-GTP, while 20S+ particles still accumulated inside the nucleus. (B) Nuclear and cytosolic fractions were separated and immunoblotted with anti-Î±7 as in Figure 5C, comparing normal nuclei with the addition of 2 mM GTPÎ³S after 1 h of assembly. Both reactions were fractionated after 20 min of incubation. The inhibition of GTPase functions after nuclear assembly did not affect the nuclear/cytoplasmic ratio of the Î±7 proteasomal marker.
	FIGURE 7:. Reconstitution of an import-compatible form of the proteasome. (A) The purified + fraction was fluorescently labeled and tested in an import assay with reconstituted nuclei, as in Figure 6. The labeled + fraction accumulated inside nuclei (right). The addition of RanQ69L-GTP blocked the import of TRITC-NLS-BSA cargo, but did not affect the nuclear accumulation of the + fraction. (B) Preincubation of labeled proteasome particles with cytosol prior to their addition into nuclear reconstitution reactions did not affect their targeting properties. The 20S+ particles were efficiently imported (top row), while 20S particles remained perinuclear (middle row) in the majority of nuclei. By contrast, when labeled 20S particles were preincubated with the unlabeled + fraction, about half of the nuclei exhibited substantial intranuclear accumulation together with nuclear rim staining (bottom row). The histogram on the right compiles the results of three separate experiments, in which individual nuclei were scored as showing either nuclear rim or rim + intranuclear staining. Labeled 20S particles were preincubated with cytosol (control) or with cytosol supplemented with the concentrated + fraction (reconstitution). Error bars represent the SE. Scale bars, 10 Î¼m.
	FIGURE 8:. Different 19S subunits show distinct nuclear targeting properties. (A) Normal (functional) nuclei and BAPTA-inhibited (poreless) nuclei were immunostained with anti-Î±7 and anti-Rpt5 and analyzed by confocal microscopy. Differential interference contrast (DIC) images and confocal midsections are shown for all nuclei. The 20S CP subunit Î±7 was missing from poreless nuclei, but anti-Rpt5 strongly stained these nuclear assembly intermediates. (B) Nuclear and cytosolic fractions were separated and compared between normal and BAPTA-inhibited nuclei, as in Figure 2B. Immunoblotting of the separate fractions confirmed that Rpt5 was present in the nuclear fraction in both normal and poreless nuclei, while Î±7 and Rpn2 only associated with normal nuclei.

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