Transportin Regulates Major Mitotic Assembly Events: From Spindle to Nuclear Pore Assembly

Abstract

Mitosis in higher eukaryotes is marked by the sequential assembly of two massive structures: the mitotic spindle and the nucleus. Nuclear assembly itself requires the precise formation of both nuclear membranes and nuclear pore complexes. Previously, importin alpha/beta and RanGTP were shown to act as dueling regulators to ensure that these assembly processes occur only in the vicinity of the mitotic chromosomes. We now find that the distantly related karyopherin, transportin, negatively regulates nuclear envelope fusion and nuclear pore assembly in Xenopus egg extracts. We show that transportin-and importin beta-initiate their regulation as early as the first known step of nuclear pore assembly: recruitment of the critical pore-targeting nucleoporin ELYS/MEL-28 to chromatin. Indeed, each karyopherin can interact directly with ELYS. We further define the nucleoporin subunit targets for transportin and importin beta and find them to be largely the same: ELYS, the Nup107/160 complex, Nup53, and the FG nucleoporins. Equally importantly, we find that transportin negatively regulates mitotic spindle assembly. These negative regulatory events are counteracted by RanGTP. We conclude that the interplay of the two negative regulators, transportin and importin beta, along with the positive regulator RanGTP, allows precise choreography of multiple cell cycle assembly events. INTRODUCTIONDuring mitosis, the intracellular architecture of the higher eukaryotic cell undergoes constant and dramatic change. The chromosomes condense, the nuclear envelope breaks down, a mitotic spindle forms and separates the chromosomes, and finally a nuclear envelope is reassembled around each set of daughter chromosomes. The movement of the cell cycle from one stage of mitosis to the next involves the coordinated action of multiple kinases and phosphatases. Recently, however, new regulators have been identified that contribute not to the timing but to the spatial control of assembly of both the mitotic spindle and the nucleus: karyopherins and RanGTP.Karyopherins are most well known for their function in interphase as the transport receptors for nuclear import and export (reviewed in Tran and Wente, 2006;Stewart, 2007). Nuclear proteins larger than ϳ20 -40 kDa are actively imported, whereas snRNAs, tRNAs, and other cargoes are exported through the nuclear pores by karyopherins.Karyopherins consist of a large family of importins (import receptors) and exportins (export receptors; reviewed in Weis, 2003;Pemberton and Paschal, 2005). The first and most studied import receptor of the karyopherin family was importin beta (Adam and Adam, 1994;Chi et al., 1995;Gorlich et al., 1995;Imamoto et al., 1995;Radu et al., 1995;Floer et al., 1997). Importin beta mediates the nuclear import of many proteins through the use of a specific adaptor, such as importin alpha (reviewed in Cook et al., 2007). Importin alpha recognizes cargoes containing classical or bipartite nuclear localization signals (NLSs), which are composed largely of basic amino acids (reviewed ...

Localization of retinitis pigmentosa 2 to cilia is regulated by Importin β2

“…In this process, Importin proteins bind and inhibit factors such as HURP and TPX2, which are essential for microtubule spindle growth, and this inhibition is relieved by Ran-GTP Nachury et al, 2001;Wiese et al, 2001;Silljé et al, 2006). Although these roles have been ascribed for Importin 1 and its Importin- binding partners, Importin 2 might also have a role in mitotic spindle assembly (Lau et al, 2009). In this report and other recent reports, we also suggest a further role for Importin proteins in centrosomal and cilia targeting (Dishinger et al, 2010;Fan et al, 2007).…”

The discovery of a Werner helicase interacting protein (WHIP) association with the nuclear pore complex

“…In Figure 4C, we probed for the presence of ELYS in our Nup107-160 immunoprecipitation to compare to published reports. [25][26][27] We found ELYS at 6 h and 9 h time points corresponding to S phase transition and the onset of mitosis (Fig. 4C).…”

“…[19][20][21][22][23][24] An additional non-Nup member may be ELYS, a putative transcription factor, which was also discovered to copurify with the Nup107-160 complex in Xenopus interphase extracts, Xenopus mitotic extracts and human cell extracts. [25][26][27] Positioned at the curvatures of the membrane embedding the NPC, the Nup107-160 subcomplex acts to stabilize these bends in the membrane. 28 This subcomplex has been classified as a keystone of NPC assembly and is required for assembly of a subset of Nups into the NPC.…”

A dominant-negative form of POM121 binds chromatin and disrupts the two separate modes of nuclear pore assembly

“…We and others have previously shown that importin-b negatively regulates several distinct steps in postmitotic NPC assembly, including chromatin seeding by ELYS-Nup107-160 (Harel et al, 2003a;Lau et al, 2009;Rotem et al, 2009;Walther et al, 2003). We next investigated whether importin-b was also capable of regulating the binding of POM121 DN to chromatin.…”