title: Nuclear import of an intact preassembled proteasome particle.
abstract: 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.
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Anca F
authors.initials: AF
authors.lastname: Savulescu
authors.forename: Hagai
authors.initials: H
authors.lastname: Shorer
authors.forename: Oded
authors.initials: O
authors.lastname: Kleifeld
authors.forename: Ilana
authors.initials: I
authors.lastname: Cohen
authors.forename: Rita
authors.initials: R
authors.lastname: Gruber
authors.forename: Michael H
authors.initials: MH
authors.lastname: Glickman
authors.forename: Amnon
authors.initials: A
authors.lastname: Harel
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D009685:Nuclear Envelope / Q000378:metabolism / Q000648:ultrastructure; D022022:Nuclear Pore / Q000378:metabolism / Q000648:ultrastructure; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D020931:ran GTP-Binding Protein