Vertebrate nuclear assembly is a complex process involving the sequential recruitment of specific proteins and membranes to chromatin. At the end of mitosis, membrane vesicles and/or ER membrane sheets arrive at the chromatin surface to fuse and form a unique structure consisting of two complete, encircling membrane bilayers [1, 2]. As soon as regions of double membrane form at the chromatin surface, nuclear pore complexes form within those regions perforating the membranes. Nuclear pore complexes span the bilayers and control virtually all traffic between the nucleus and cytoplasm [3, 4]. The 125-megadalton vertebrate nuclear pore is composed of multiple copies of ~30 different nucleoporins, only three of which are integral membrane proteins . The majority of nucleoporins are recruited from soluble cytoplasmic subunits. The assembly of these nucleoporins into the 500–1000 protein complex is a daunting task, as nucleoporins must sequentially and precisely assemble in the correct order and location [6–8]. Determining the choreographed molecular mechanism by which nucleoporins assemble into functional pores within the double nuclear membranes is a matter of intense research.
The nuclear import factor, importin beta, and its regulatory counterpart, the small GTPase Ran, were revealed to be two key regulatory factors controlling this choreography, both for nuclear membrane fusion and separately for nuclear pore assembly [9–13]. Addition of excess human importin beta to a Xenopus nuclear reconstitution system disrupts the endogenous ratio between importin beta and RanGTP. This disruption blocks proper nuclear membrane fusion and the subsequent step of nuclear pore assembly [9, 10]. The block to nuclear membrane fusion was found to be reversible by the positive regulator, RanGTP, but the block to pore assembly, oddly, was not [9, 10]. There is, however, much precedence for positive Ran effects on nuclear pore assembly: The addition of RanQ69L, a Ran mutant constitutively in the GTP-bound state, to the Xenopus reconstitution system causes greatly increased nuclear pore assembly and ectopic formation of additional pores in cytoplasmic membranes or annulate lamellae [9, 10, 14–17]. These studies led to the hypothesis that importin beta acts in the cell cycle to negatively regulate nuclear pore formation and that it does so by binding to nucleoporins, preventing them from interacting with one another. When such importin beta/nucleoporin complexes enter the vicinity of high RanGTP, importin beta preferentially binds RanGTP, releasing its hold on the nucleoporins. A high concentration of RanGTP is produced only around chromatin, due to the chromosomal localization of the RanGEF, RCC1 [18–21]. The freed nucleoporins are then able to interact with one another in the correct location and the correct ratio to form nuclear pores at the chromatin periphery [9, 10, 22].
Prior to the discovery of its role as a negative regulator of nuclear membrane fusion and pore assembly, importin beta was elegantly shown by a number of groups to be a negative regulator of mitotic spindle assembly in Xenopus laevis egg extract [23–29], mammalian cell lines [25, 30], Drosophila Melanogaster , and Caenorhabditis elegans  (Reviewed in [11, 12, 33, 34]). In this arena, mitotic spindle assembly factors (SAFs) such as TPX2, NuMa, and XCTK2 are found to be imported into the nucleus by importin beta and localize there throughout interphase in Xenopus egg extract [27, 28, 35–37] and mammalian cell lines [35, 38] (Reviewed in [39–41]). This sequestration effectively prevents the SAFs from interfering with interphase microtubule formation in the cytoplasm. At mitosis when the nuclear envelope breaks down, the SAFs are released from the nucleus and come under importin beta regulation. Binding of importin beta inhibits the SAFs throughout the cell, except in the vicinity of the RanGTP-rich chromosomes. There, importin beta preferentially binds to RanGTP, releasing its hold on the spindle assembly factors and allowing them to initiate mitotic spindle formation around the chromosomes.
These nuclear and spindle assembly studies on the regulatory role of importin beta were performed in interphase and mitotic assembly systems derived from Xenopus eggs [23, 26–28, 35, 42–50]. In a Xenopus interphase egg extract, nuclei normally assemble spontaneously around added chromatin or DNA [51–60]. In contrast, in a Xenopus mitotic egg extract, spindles spontaneously form around the added chromatin [61, 62]. Thus, these in vitro systems are powerful tools for studying both nuclear and mitotic spindle assembly.
Upon further analysis, we realized that the recombinant importin beta used in all the Xenopus studies of nuclear and spindle assembly was, in actuality, human importin beta [9, 10, 25, 27–30, 37, 63–68]. (Xenopus importin beta had neither been identified nor cloned and thus was not available for the studies). The use of recombinant human importin beta in the Xenopus system led to a further key question: Is importin beta an authentic negative regulator of cellular function, or does human importin beta act as a dominant negative mutant as a result of sequence variation between the human and Xenopus proteins?
To address this question, in this study we identified, cloned, and tested recombinant Xenopus importin beta for its role in nuclear membrane fusion and nuclear pore assembly. We found Xenopus importin beta to act identically to human importin beta, i.e., it acts as a negative regulator of both nuclear membrane fusion and pore assembly, finally validating the conclusion that importin beta is an authentic negative regulator of cell cycle steps. However, in examining tagged importin betas, which include the form that has been used in all the previous studies, we found evidence that the tag renders importin beta mutant in its response to Ran, but does so specifically with respect to pore assembly. This impairment of importin beta raises interesting hypotheses as to why nuclear pore assembly is unique, which will be discussed here.