Importin β Negatively Regulates Nuclear Membrane Fusion and Nuclear Pore Complex Assembly

Abstract

Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. We report that importin ␤ negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin ␤ is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin ␤ down-regulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellae-like structures. We propose that a precise balance of importin ␤ to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin ␤ 45-462 allows membrane fusion but produces nuclei lacking any NPCs. This reveals distinct importin ␤-regulation of NPC assembly. Excess full-length importin ␤ and ␤ 45-462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin ␤ NPC block, which maps downstream of GTP␥S and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 M RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin ␤. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly. INTRODUCTIONIn cells from yeast to mammals, importin ␣ and ␤ act together to ferry classical nuclear localization signal (NLS)-bearing proteins into the nucleus (Gorlich and Kutay, 1999;Stoffler et al., 1999;Damelin and Silver, 2000;Rout et al., 2000;Conti and Izaurralde, 2001;Vasu and Forbes, 2001;Damelin et al., 2002;Weis, 2003). Once in the nucleus the small GTPase Ran binds to importin ␤, displacing importin ␣ and the NLS cargo, thus completing import. In the nucleus, Ran is kept in a GTP state by the constant action of its chromatinbound Ran-GEF, RCC1 (Melchior and Gerace, 1998;Macara, 2001;Dasso, 2002;Kalab et al., 2002;Schwoebel et al., 2002;Steggerda and Paschal, 2002). In contrast, RanGDP is the predominant form found in the cytoplasm due to the cytoplasmic localization of RanGAP.The horizons for importin ␣ and ␤ were unexpectedly broadened when they were found to play a very different role at mitosis. In metazoans, importin ␣ and ␤ are released to the cytosol by nuclear breakdown, where they act to inhibit proteins essential for mitotic spindle assembly. However, the inhibition of spindle assembly is reversed by Ran in the vicinity of mitotic chromosomes, where RanGTP continues to be produced by chromatin-bound RCC1 (Kalab et al., 1999;Gruss et al., 2001;Nachury et al., 2001;Wiese et al., 2001;Dasso, 2002). Thus, a spindle forms only around the mitotic (ER) chromosomes and not elsewhere in the cytoplasm.At the end ...

Capture of AT-rich Chromatin by ELYS Recruits POM121 and NDC1 to Initiate Nuclear Pore Assembly

“…Because Xenopus ELYS is sensitive to degradation, Xenopus egg or cell lysates were prepared for immunoblotting by heating at 60°C for 10 min in 1ϫ sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 50 mM DTT, supplemented with bromophenol blue). Other antibodies used in this study included anti-xNup160, anti-hNup133, anti-rat Nup98 GLFG (Harel et al, 2003b); anti-hNup85, anti-Xenopus POM121, anti-gp210 (Harel et al, 2003a); anti-xNup43, anti-xNup37 ; antiNup93, anti-hNup205 (Miller and Forbes, 2000); anti-Tpr ; anti-Xenopus importin ␤ (Rasala et al, 2006); anti-xNup155 (S. Vasu and D. J. Forbes, unpublished data); anti-mNup53, anti-xNDC1 (V. Delmar and D. J. Forbes, unpublished data); anti-xNup50 (R. Sekhorn, unpublished data); antiOrc2, anti-RCC1, anti-Mcm3 (generous gifts from Z. You, Washington University, St. Louis, MO); anti-FG nucleoporin antibody mAb414 (used to probe for Nup358, Nup214, Nup153, and Nup62 by immunoblot) and anti-GST (Covance, Berkeley, CA); anti-human importin ␣ (BD Transduction Laboratories, Lexington, KY), anti-GAPDH (Calbiochem, San Diego, CA), and antiribophorin (Serotec, Oxford, United Kingdom).…”

“…Two major mechanistic models have been proposed that differ substantially in regard to the role of the nuclear membranes in this process. One model, and considerable data, proposes that NPCs assemble within patches of double nuclear membranes as soon as those membranes begin to form on the surface of chromatin in late anaphase (Macaulay and Forbes, 1996;Goldberg et al, 1997;Harel et al, 2003a;Anderson and Hetzer, 2007;Baur et al, 2007;M. Hetzer, personal communication).…”

The nuclear pore complex – structure and function at a glance

“…This assembly process is proposed to be initiated by the targeting of the Y-complex to chromatin, which is facilitated by small-DNAbinding motifs (AT hooks) that are predicted to be present in ELYS (Rasala et al, 2008). As with NPC assembly during interphase, Ran -as well as its effectors -and importin-b regulate Nup recruitment to chromatin (Harel et al, 2003;Walther et al, 2003). However, once ELYS is localized to chromatin, assembly continues with the remainder of the Nup107 subcomplex, which interacts with the TM-Nups Ndc1 and Pom121 (Dultz et al, 2008;Rasala et al, 2008).…”

Assembly of nuclear pore complexes mediated by major vault protein

“…Previous studies on separating membrane-related events from NPC assembly relied on the use of chemical inhibitors such as the calcium chelator BAPTA or on the depletion or enrichment of proteins that are positively (RanGTP) or negatively (importin-β) involved in NPC assembly (Macaulay and Forbes, 1996;Harel et al, 2003;D'Angelo et al, 2006). Our in vitro system described here is different as it solely depends on the physical separation of two membrane populations.…”