InteractiveFly: GeneBrief

karyopherin α1: Biological Overview | References

Gene name - karyopherin α1

Synonyms - importin alpha1

Cytological map position - 76C6-76C6

Function - signaling

Keywords - nucleo-cytoplasmic transport, spermatogenesis

Symbol - Kap-α1

FlyBase ID: FBgn0024889

Genetic map position - 3L:19,800,347..19,803,498 [+]

Classification - Karyopherin (importin) alpha

Cellular location - cytoplasmic and nuclear

NCBI links: | EntrezGene
Recent literature
Wang, S., Lu, Y., Yin, M. X., Wang, C., Wu, W., Li, J., Wu, W., Ge, L., Hu, L., Zhao, Y. and Zhang, L. (2016). Importin α1 mediates Yorkie nuclear import via an N-terminal non-canonical nuclear localization signal. J Biol Chem 291: 7926-7937. PubMed ID: 26887950
The Hippo signaling pathway controls organ size by orchestrating cell proliferation and apoptosis. When the Hippo pathway is inactivated, the transcriptional co-activator Yorkie translocates into the nucleus and forms a complex with transcription factor Scalloped to promote the expression of Hippo pathway target genes. Therefore, the nuclear translocation of Yorkie is a critical step in Hippo signaling. This study provides evidence that the N-terminal 1-55 amino acids of Yorkie, especially Arg-15, were essential for its nuclear localization. By mass spectrometry and biochemical analyses, it was found that Importin α1 can directly interact with the Yorkie N terminus and drive Yorkie into the nucleus. Further experiments show that the upstream component Hippo can inhibit Importin α1-mediated Yorkie nuclear import. Taken together, this study has identified a potential nuclear localization signal at the N-terminal end of Yorkie as well as a critical role for Importin alpha1 in Yorkie nuclear import.
Yashiro, R., Murota, Y., Nishida, K. M., Yamashiro, H., Fujii, K., Ogai, A., Yamanaka, S., Negishi, L., Siomi, H. and Siomi, M. C. (2018). Piwi nuclear localization and its regulatory mechanism in Drosophila ovarian somatic cells. Cell Rep 23(12): 3647-3657. PubMed ID: 29925005
In Drosophila ovarian somatic cells (OSCs), Piwi represses transposons transcriptionally to maintain genome integrity. Piwi nuclear localization requires the N terminus and PIWI-interacting RNA (piRNA) loading of Piwi. However, the underlying mechanism remains unknown. This study shows that Importinalpha (Impalpha) plays a pivotal role in Piwi nuclear localization and that Piwi has a bipartite nuclear localization signal (NLS). Impalpha2 and Impalpha3 are highly expressed in OSCs, whereas Impalpha1 is the least expressed. Loss of Impalpha2 or Impalpha3 forces Piwi to be cytoplasmic, which is rectified by overexpression of any Impalpha members. Extension of Piwi-NLS with an additional Piwi-NLS leads Piwi to be imported to the nucleus in a piRNA-independent manner, whereas replacement of Piwi-NLS with SV40-NLS fails. Limited proteolysis analysis suggests that piRNA loading onto Piwi triggers conformational change, exposing the N terminus to the environment. These results suggest that Piwi autoregulates its nuclear localization by exposing the NLS to Impalpha upon piRNA loading.
Beckmann, J. F., Sharma, G. D., Mendez, L., Chen, H. and Hochstrasser, M. (2019). The Wolbachia cytoplasmic incompatibility enzyme CidB targets nuclear import and protamine-histone exchange factors. Elife 8. PubMed ID: 31774393
Intracellular Wolbachia bacteria manipulate arthropod reproduction to promote their own inheritance. The most prevalent mechanism, cytoplasmic incompatibility (CI), traces to a Wolbachia deubiquitylase, CidB, and CidA. CidB has properties of a toxin, while CidA binds CidB and rescues embryonic viability. CidB is also toxic to yeast where both host effects and high-copy suppressors of toxicity were identified. The strongest suppressor was karyopherin-alpha, a nuclear-import receptor; this required nuclear localization-signal binding. A protein-interaction screen of Drosophila extracts using a substrate-trapping catalytic mutant, CidB*, also identified karyopherin-alpha; the P32 protamine-histone exchange factor bound as well. When CidB* bound CidA, these host protein interactions disappeared. These associations would place CidB at the zygotic male pronucleus where CI defects first manifest. Overexpression of karyopherin-alpha, P32, or CidA in female flies suppressed CI. It is proposed that CidB targets nuclear-protein import and protamine-histone exchange and that CidA rescues embryos by restricting CidB access to its targets.

Importin αs mediate nuclear transport by linking nuclear localization signal (NLS)-containing proteins to importin beta1. Animal genomes encode three conserved groups of importin α's, α1's, α2's (Pendulin), and α3's, each of which are competent to bind classical NLS sequences. Using Drosophila melanogaster, the isolation and phenotypic characterization of the first animal importin α1 mutant is described. Animal α1's are more similar to ancestral plant and fungal α1-like genes than to animal α2 and α3 genes. Male and female importin α1 (Dα1) null flies developed normally to adulthood (with a minor wing defect) but were sterile with defects in gametogenesis. The Dα1 mutant phenotypes were rescued by Dα1 transgenes, but not by Dα2 or Dα3 transgenes. Genetic interactions between the ectopic expression of Dα1 and the karyopherins CAS and importin β1 suggest that high nuclear levels of Dα1 are deleterious. It is concluded that Dα1 performs paralog-specific activities that are essential for gametogenesis and that regulation of subcellular Dα1 localization may affect cell fate decisions. The initial expansion and specialization of the animal importin α-gene family may have been driven by the specialized needs of gametogenesis. These results provide a framework for studies of the more complex mammalian importin α-gene family (Ratan, 2008).

The animal importin α-gene family is diverse, having undergone multiple rounds of duplications and lineage-specific expansions. Most animal importin α's belong to one of three conserved clades, referred to here as α1, α2, and α3. Animal α1 genes are more similar to plant and fungal α1-like genes than to animal α2 or α3 genes, which arose from an animal α1-like progenitor to perform specialized roles in animal development and differentiation (Goldfarb, 2004). The evolution and maintenance of α1, α2, and α3 genes among animals is likely due to their specialized roles in conserved aspects of animal development. Although importin α1, α2, and α3 proteins are coexpressed in many adult tissues, they exhibit complex temporal and spatial expression patterns during development (see Kamei, 1999; Hogarth, 2006). Animal importin α1's, α2's, and α3's all mediate the import of classical NLS-containing cargoes and, in addition, each paralog is specialized to bind and mediate the import of distinct repertoires of NLS cargoes. Importin α's may also mediate the import of a few cargoes independent of β1. Finally, importin α's play specialized roles in cell processes other than nuclear transport (Ratan, 2008).

The proliferation of the importin α-gene family in animals may have been driven in part by the specialized needs of gametogenesis (Goldfarb, 2004; Hogarth, 2005). Importin α1, α2, and α3 proteins are differentially expressed during spermatogenesis in both Drosophila (Giarre, 2002) and mouse (Hogarth, 2006). Two of the three Caenorhabditis elegans importin α's are expressed exclusively in the germline and are both required for gametogenesis (Geles, 2001; Geles, 2002). Likewise, Drosophila lacking importin α2 (Dα2) develop normally to adulthood but have serious defects in gametogenesis (Gorjanacz, 2002; Mason, 2002; Mason, 2003). The spermatogenesis defect of Dα2 null flies is due to the loss of an activity shared by all three paralogs, since Dα1, Dα2, and Dα3 transgenes rescued the defect. In contrast, the role of Dα2 in oogenesis is unique since only Dα2 transgenes could rescue the phenotype. Like the C. elegans importin α3, Dα3 is required for somatic development and differentiation (Mason, 2003). Dα3 may also be required for gametogenesis but mutant animals die as larvae. Therefore, in addition to shared housekeeping roles in classical nuclear transport, importin α2's and α3's each have unique roles in animal-specific processes such as gametogenesis. What remains is to describe the consequences of mutating the single Drosophila importin α1 (Dα1) (Ratan, 2008).

This paper describes the isolation and characterization of a Dα1 null mutation. Like Dα2 mutant flies, Dα1 null flies develop to adulthood with severe defects in gametogenesis. Spermatogenesis in Dα1 null flies is arrested and males are completely sterile. Oogenesis is morphologically less severely affected, but virtually all Dα1 null females are sterile. In addition, overexpression of Dα1 results in defects in tergite development and viability that are enhanced by mutations in the importin α-recycling factor CAS and suppressed by mutations in importin β. This is the first genetic analysis of an animal importin α1 mutant and completes an analysis on the null phenotypes of the conventional Drosophila α1, α2, and α3 gene family (Ratan, 2008).

Cargo adapters such as importin α may have evolved to provide a greater range of control over nuclear transport in response to variable environmental conditions. The evolution of multiple importin α-genes would seem to extend the utility of these adapters by allowing the independent control of distinct sets of cargo repertoires. A genetic approach was taken in Drosophila to analyze the in vivo function of the conserved family of animal importin α1's, α2's, and α3's. In addition to binding unique repertoires of NLS cargoes, all three types likely share housekeeping duties in cNLS cargo import. The contribution of individual importin α's to redundant activities is influenced by their differing temporal and spatial expression patterns in various cells and tissues. In this study the first animal importin α1 mutant is described (Ratan, 2008).

The key finding here is that Dα1 mutant flies develop (almost) normally to adulthood but both males and females are sterile due to defects in gametogenesis. Dα1 null flies also exhibit a minor wing defect, so Dα1's nonredundant activities extend in this small way to somatic development. In contrast to Dα1 and Dα2, Dα3 is required for somatic development and Dα3 mutants arrest as larvae. Interestingly, Dα1 and Dα2 mutants display distinct phenotypes in gametogenesis. Spermatogenesis is more severely affected than oogenesis in Dα1 mutants, while Dα2 mutants have more severe defects in oogenesis (Mason, 2002). Dα2 is not absolutely essential for spermatogenesis -- some motile sperm and viable progeny are produced by mutant males -- and the defect can be rescued by Dα1, Dα2, or Dα3 transgenes. In contrast, no viable sperm are produced in Dα1 mutants, and only a Dα1 transgene can rescue the defect. Therefore, Dα1 serves a paralog-specific role in spermatogenesis that is distinct from the role of Dα2 in this process (Ratan, 2008).

Dα1 and Dα2 are both required for gametogenesis and have no significant roles in somatic development. It seems likely, therefore, that the evolutionary expansion of the importin α-gene family occurred to serve the uniquely complex processes of spermatogenesis and oogenesis, both of which involve the differentiation of germ-line stem cells using analogous signaling pathways (Gilboa, 2004). Dα1 plays an especially important paralog-specific role in spermatogenesis. All three importin α's are expressed in the fly testes, although in distinct, partially overlapping patterns that correspond to different stages of spermatogenesis, which include stem cell division, spermatogonial divisions, growth, meiotic divisions, and spermatid differentiation (reviewed in Gilboa, 2004; Hogarth, 2005). The expression of Dα1 overlaps with Dα2 expression during meiosis, and later with Dα3 during differentiation and individualization (Giarre, 2002). Dα1 is expressed at low levels in testes until the growth stage, when it appears cytoplasmic. Dα1 levels rise during meiosis when it accumulates in spermatid nuclei. Dα1 levels are lower during differentiation and, by the time spermatid heads become aligned toward the wall of the testes, are equally distributed between the nucleus and cytoplasm. Dα1 was not detectable in sperm with elongated heads. The defects exhibited by Dα1 and Dα2 mutants are manifested at different stages of sperm differentiation, although the timing and nature of these defects do not necessarily correspond to when and where during spermatogenesis these factors are actually required (Ratan, 2008).

The oogenesis defects of Dα1 and Dα2 mutant flies are also distinct from one another, and both phenotypes are due to paralog-specific activities. The cause of the severe Dα2 mutant phenotype (deflated oocytes) is likely related to the Dα2-dependent targeting of Kelch to ring canals, through which nurse cell cytoplasm is dumped into the developing oocyte (Gorjanacz, 2002). Kelch localization and dumping appear normal in Dα1 mutant females. Giarre (2002) reported that Dα1 expression in ovaries is weaker than that of Dα2 or Dα3, and is, therefore, unlikely to play a major role. This prediction is partially supported by the finding that the ovaries and eggs of Dα1 null flies are only mildly defective. Still, Dα1 must have an important role in oogenesis since almost all mutant females are sterile. It remains possible that the female sterility is due to a behavioral phenotype in egg laying or mating or some other defect that was too subtle to notice (Ratan, 2008).

The finding that two of the three conventional Drosophila importin α's are specialized to serve important roles in gametogenesis has a strong parallel in C. elegans (reviewed in Goldfarb, 2004; Hogarth, 2005). The C. elegans genome encodes three importin α's, IMA-1, IMA-2, and IMA-3, two of which (IMA-1 and IMA-2) localize exclusively to the germ line and are required for gametogenesis (Geles, 2001; Geles, 2002). Therefore, two of the importin α's in both fly and worm are required for gametogenesis. IMA-3, a conventional α3 type, is expressed in both somatic and germ-line cells, and like Dα3, is required for somatic development (Geles, 2001). Although IMA-1 and IMA-2 are highly divergent and dissimilar to any of the conventional types, their exclusive expression in the germ line and important role in gametogenesis suggest they may be functional homologs of Dα1 and Dα2. Also, like Dα2, IMA-2 displays cell cycle-dependent shifts between the nucleus and cytoplasm in the gonads, and both accumulate around chromosomes at the onset of nuclear envelope breakdown (Geles, 2002). Taken together, these results suggest the possibility that the special needs of gametogenesis may have driven the early expansion and specialization of the metazoan animal importin α-gene family. The complex temporal expression patterns of the five mouse importin α1's, α2's, and α3's in testes indicate that this role likely extends to mammalian spermatogenesis, which, in many ways, is similar to spermatogenesis in flies (Ratan, 2008).

Because importin α1's are very similar both by sequence and gene structure to ancestral plant and fungal α1-like genes, it was originally expected that the loss of Dα1 would cause defects in the nuclear transport of many important proteins with catastrophic consequences. Therefore, it was initially surprising to find that Dα1 null flies developed normally to adulthood with only a slight wing defect. Phenotypically, then, Dα1 is more similar to Dα2, whose loss also primarily affects gametogenesis. At gene structure and primary sequence levels Dα2 is more similar to Dα3. Thus the evolutionary history of the three genes does not predict the nature of their mutant phenotypes. It is hypothesized that the ancient and essential role the importin α's play in cNLS cargo import is redundantly supported in somatic tissues by the partially overlapping coexpression of the three paralogous proteins. The loss of any one is apparently masked by the activity of one or both of the others. Most of the phenotypes that appear in single gene mutants are likely due to paralog-specific functions that were divided among the genes following the duplications that gave rise to the extant importin α-gene family. An exception is the spermatogenesis defect of Dα2 mutant flies that is rescued by any of the three paralogs (Mason, 2002). It is established that importin α1's each have both shared and distinct cargo repertoires. The simplest explanations for the paralog-specific phenotypes associated with Dα1, Dα2, and Dα3 mutants invoke deficiencies in the nuclear import of their distinct NLS cargoes (Ratan, 2008).

The genetic interactions between coectopic expression of Dα1 and Dcas and Ketel are consistent with the idea that the tergite defects and lethality are the result of increases in the levels of importin α in nuclei. Genetic manipulations that would be expected to decrease nuclear levels of Dα1 (overexpression of Dcas or loss-of-function Ketel mutants) mitigated the effects of overexpressing Dα1. Likewise, manipulations that would be expected to increase nuclear levels of Dα1 (overexpression of Ketel or loss-of-function Dcas mutants) enhanced Dα1 overexpression phenotypes. Interestingly, an increase in cNLS cargo levels also enhanced the Dα1 overexpression defects. Here, higher cNLS cargo levels could be expected to recruit more Dα1 into targeting complexes with importin β1 (Ketel), resulting in higher steady state nuclear levels of Dα1. Taken together, these results argue that higher than normal nuclear levels of Dα1 are deleterious, and that the nucleocytoplasmic trafficking of nuclear transport factors must be carefully balanced during development (Ratan, 2008).

The defect in tergite development observed in Dα1-overexpressing flies may lend insight into the mechanisms underlying the deleterious effects of excess nuclear importin α. Development of the tergites involves a tightly coordinated process of epithelial cell sheet replacement during which the adult tergites arise from histoblast nests that proliferate and spread to replace larval epidermal cells during pupal morphogenesis. The tergite defects observed in Dα1-expressing abdomens may be attributable either to the failure of the adult histoblast nests to proliferate or spread correctly or to a failure of the larval epidermal cells to undergo apoptosis since both of these processes are thought to be codependent. The genetic interactions between Dcas and Dα1 may be especially relevant to understanding the tergite phenotypes associated with Dα1 overexpression. Expression of CAS antisense RNA in MCF-7 breast carcinoma cells, which likely leads to increased nuclear levels of importin α, inhibits apoptosis. It is possible, then, that elevated levels of nuclear importin α inhibit apoptosis in these cells. By analogy, it is possible that elevated levels of nuclear Dα1 interfere with the apoptosis of larval epidermal cells, the persistence of which might impair the ability of the adult cuticle to properly proliferate and spread. Consistent with this hypothesis, blocking cell death in the larval epidermal cells of the abdomen result in defects in spreading of the histoblast nests and resulted clefts in the abdominal cuticle. Alternatively, these tergite phenotypes may be caused by defects in tergite development since thermocautery of histoblast nests also produces similar tergite defects. Nonetheless, it is intriguing to speculate that the regulated subcellular localization of importin α-proteins affects susceptibility to proapoptotic signals (Ratan, 2008).

This analysis of Dα1 complements a previous analyses of Dα2 and Dα3 (Mason, 2002; Mason, 2003). It is now possible to now say that two of the three conserved Drosophila importin α-genes are required almost exclusively for gametogenesis (Dα1 and Dα2), and only one (Dα3) is required for general viability. The larger picture emerges of a gene family that likely arose by gene duplication to serve the newly evolving requirements of gametogenesis. Following their initial establishment, each of the three paralogous genes was available to evolve specialized (derived) roles and, in mammals, undergo further gene duplications and specializations. It is curious that Dα1, which is more similar to ancient plant and fungal importin α1-like genes than to Dα2 or Dα3, exhibits paralog-specific phenotypes only in derived processes such as gametogenesis and wing development. It is hypothesized that α1 genes are not functionally constrained; rather, ancestral α2/α3 genes simply diverged. Why ancestral α2 and α3 genes evolved more rapidly remains a mystery, although important clues no doubt lie among their largely unexplored NLS cargo repertoires. It will be extremely interesting to learn if these roles and relationships are conserved in the more complex mouse and human importin α-gene family (Ratan, 2008).

Regulation of Greatwall kinase by protein stabilization and nuclear localization

Greatwall (Gwl) functions as an essential mitotic kinase by antagonizing protein phosphatase 2A. This study identified Hsp90, Cdc37 and members of the importin alpha and beta families as the major binding partners of Gwl. Both Hsp90/Cdc37 chaperone and importin complexes associated with the N-terminal kinase domain of Gwl, whereas an intact glycine-rich loop at the N-terminus of Gwl was essential for binding of Hsp90/Cdc37 but not importins. Hsp90 inhibition led to destabilization of Gwl, a mechanism that may partially contribute to the emerging role of Hsp90 in cell cycle progression and the anti-proliferative potential of Hsp90 inhibition. Moreover, in agreement with its importin association, Gwl exhibited nuclear localization in interphase Xenopus S3 cells, and dynamic nucleocytoplasmic distribution during mitosis. KR456/457 was identified as the locus of importin binding and the functional NLS of Gwl. Mutation of this site resulted in exclusion of Gwl from the nucleus. Finally, this study showed that the Gwl nuclear localization is indispensable for the biochemical function of Gwl in promoting mitotic entry (Yamamoto, 2014). Entry into mitosis requires activation of maturation-promoting factor (MPF), a complex of cyclin B and Cdk1. During mitotic entry, MPF activation is triggered by Cdc25-mediated dephosphorylation of Cdk1. In addition to mechanisms that directly control the activity of Cdc25 and MPF, dynamic and regulated nucleocytoplasmic localization of these factors was also demonstrated to be important. In particular, both cyclin B and Cdc25 need to localize to the nucleus to trigger mitotic entry. It has been shown that phosphorylation of cyclin B by Polo-like kinase 1 (Plk1) promotes its nuclear localization. Furthermore, checkpoint kinases, Chk1 and Chk2, phosphorylate Cdc25, and thereby creating a binding site for 14-3-3 proteins which sequestrate Cdc25 in cytoplasm and inhibit M-phase entry. In addition to MPF, another enzyme required for both mitotic entry and maintenance is the Greatwall kinase (Gwl). Recent evidence revealed that Gwl inhibits the activity of protein phosphatase 2A/B55δ, the principal phosphatase acting on Cdk-phosphorylated substrates (Yamamoto, 2014).

The crucial nature of this inhibition was demonstrated by the failure of M-phase maintenance in egg extracts with full activity of MPF and other mitotic kinases but deficient of Gwl function. On the other hand, the presence of Gwl greatly reduced the amount of MPF required for mitotic entry (Yamamoto, 2014).

The mechanism of PP2A/B55δ inhibition by Gwl has been nicely solved with the recent identification of Endosulfine and its related family member, cAMP-regulated phosphoprotein 19 kDa, as substrates of Gwl. These substrates specifically bind to and inhibit PP2A/B55δ when they are phosphorylated by Gwl. While compelling evidence obtained in a variety of experimental systems revealed an essential role of Gwl in mitotic regulation, very little has been learned about how Gwl itself is regulated. It has been shown that Gwl is phosphorylated in M-phase, in quantitative correlation with its kinase activity (Yamamoto, 2014).

Mitotic phosphorylation of Gwl by MPF within its presumptive activation loop is required for Gwl activation, suggesting that MPF acts upstream of Gwl during mitotic entry. It has also been shown that Gwl can be phosphorylated by Plk1, whereas the specific function and regulation of this modification remains to be further clarified (Yamamoto, 2014).

In the search for new regulators of Gwl, a proteomic analysis revealed 2 groups of proteins as the major binding partners of Gwl which were co-purified with Gwl from G2 and M phase Xenopus oocytes. Specifically, Gwl bound both the chaperone complex Hsp90/Cdc37 and importin α/β through its N-terminus. Hsp90 was required to stabilize Gwl in both interphase and M-phase extracts. Furthermore, this study identified the functional NLS in Gwl that mediated its binding to importins. Mutation of amino acid residues KR456/457 within the NLS led to exclusively cytoplasmic localization of the protein in Xenopus S3 cells and interfered its function in promoting mitotic entry in egg extracts (Yamamoto, 2014).

Several heat shock proteins function as molecular chaperones. In particular, Hsp90 has been related to regulators of the cell cycle, including Cdk1, p53, Cdk4, and Plk1. It has been shown that the evolutionarily conserved protein association between Plk1 and Hsp90 was required for the stability of Plk1, and thereby involved in centrosomal functions and metaphase-to-anaphase transition (Yamamoto, 2014).

This study discovered Hsp90 as a major associated partner for Gwl. Further analysis showed that the N-terminal segment of Gwl mediated its association with Hsp90. Interestingly, a glycine-rich loop in the N-terminal Gwl was required for Hsp90 association, and a G41S mutant form of Gwl was deficient in Hsp90 association. Proteomic identification of Gwl-associated proteins also revealed its association with Cdc37, the co-chaperone of Hsp90 involved in recognition of some substrates. Initially identified in budding yeast as a cell division cycle regulator, Cdc37 has been shown to interact with Cdk4, Cdk5, and several other cell cycle factors. Consistently, this study found that G41S Gwl lost the association with Cdc37, confirming the involvement of the glycine-rich loop. Importantly, inhibition of Hsp90 reduced the protein stability of Gwl and suppressed M-phase entry, suggesting that complexing to the Hsp90/Cdc37 chaperone is essential for Gwl function. The study therefore revealed Gwl as a key client protein through which Hsp90/Cdc37 chaperone regulates cell division (Yamamoto, 2014).

This new discovery contributes to better understanding of cell cycle regulation, and yields potential implication to cancer therapy. In fact, Hsp90 is frequently overexpressed in cancer cells, whereas its inhibition exhibits anti-proliferative potentials and is being explored in multiple clinical trials for treatment of various types of cancer. The proper function of cellular enzymes often involves sophisticated regulation of subcellular localization, in addition to enzymatic activity and protein stability. For instance, cyclin B/Cdk1 is imported into the nucleus before nuclear membrane breaks down to promote mitotic entry. Similarly, dynamic subcellular localization of Plk1 and Aurora A has been shown to regulate their functions during mitotic progression. One mechanism to regulate the nuclear localization of a protein is through association with the importin family members. Importin is typically composed of 2 subunits, importin α and importin β. For nuclear transport, importin β either directly binds and transports cargo proteins, or is adapted to the cargo proteins through importin α. This study reports that Gwl associates with both importin α and Importin β, suggesting that the nuclear localization of Gwl is mediated by importin. In cultured Xenopus cells, this study showed that Gwl localized to the nucleus in interphase, and was diffused into the cytoplasm in mitosis. This detailed analysis identified a functional NLS around KR456/457. Mutation of the NLS abolished the association of Gwl with importin α and Importin β, and led to Gwl nuclear exclusion and cytoplasmic sequestration (Yamamoto, 2014).

Independent studies in Drosophila and mammalian cells also characterized the nuclear localization of Gwl. The Drosophila homolog of Gwl contains 2 essential NLS motifs within the central region, mutation of which disrupted its nuclear location. However, neither of these NLS motifs in Drosophila is conserved in vertebrates. The NLS of human Gwl was found around KR444/445, the corresponding residues of KR456/457 in Xenopus Gwl. Thus, the NLS of Xenopus Gwl, as identified in this study, represents a well-conserved mechanism in vertebrate animals (Yamamoto, 2014).

Studies in Drosophila and human cells suggested a pre-mitotic transport of Gwl from nucleus to cytoplasm. It was also observed in Xenopus cells that nuclear Gwl started to diffuse into cytoplasm before centrosomes reached opposite positions. While it remained unclear how this nucleocytoplasmic translocation of Gwl may contribute to the regulation of mitotic entry, previous studies indicated that the translocation was dependent on Gwl phosphorylation by CDK1, and possibly also Plk1 (Yamamoto, 2014).

It was also suggested that the kinase activity of Gwl was required for its nuclear export as a kinase-dead form of Gwl harboring a mutation at the ATP-binding site was deficient in nuclear export. However, a different form of kinase-dead Gwl used in this study exhibited a similar pattern of nucleocytoplasmic translocation as the WT. Notably, this mutant form of Gwl was efficiently phosphorylated in mitosis, as judged by the retarded gel migration. Furthermore, it was observed that Gwl was reconcentrated into the daughter nuclei in telophase, seemingly before mitotic chromosomes are fully decondensed. By comparison, the NLS-deficient form of Gwl remained diffused in the cytoplasm during mitotic exit (Yamamoto, 2014).

An important lesson learned from previous studies on Cdk1/cyclin B, Plk1, Aurora kinases, and other mitotic regulators was that their subcellular localization often constitutes an essential mechanism to ensure the proper function of these factors. Regulation of Gwl nuclear localization is functionally important as the NLS-deficient Gwl was unable to fully rescue the mitotic defects caused by Gwl-depletion in both Drosophila and mammalian cells (Yamamoto, 2014).

To shed new light on the role of Gwl nuclear localization, whether the NLS is required for the biochemical function of Gwl was investigated. It has been shown that Gwl promotes mitotic entry through phosphorylation of Ensa/Arpp-19, which subsequently inhibit PP2A/B55δ. Interestingly, the results showed that the NLS-deficient Gwl failed to promote mitotic entry in extracts supplemented with sperm nuclei, despite that this mutant possesses intact and functional kinase domains and promoted mitotic entry in extracts devoid of nucleus formation. Notably, a number of previous studies also characterized the nuclear import of cyclin B1 and Cdc25C in Xenopus egg extracts (Yamamoto, 2014).

These studies argue that even though Xenopus egg extracts can biochemically undergo cell cycle progression in the absence of nuclei, nucleocytoplasmic regulation of the cell cycle is recapitulated in this system when sperm nuclei are reconstituted. As another example, although Xenopus egg extracts support DNA replication without nuclei, it has been shown that the NLS of cyclin E is required for DNA replication in Xenopus egg extracts with the supplementation of sperm nuclei. Moreover, the nuclear import of cyclin B1 is required for mitotic entry in Xenopus oocytes, despite that enucleated Xenopus oocytes still exhibit mitotic activation of Cdk1 (Yamamoto, 2014).

While future analyses are required to reveal how the nuclear localization of Gwl contributes to its biochemical function, a number of possibilities are proposed. First, Gwl may phosphorylate its substrates in the nucleus to promote mitotic entry. Alternatively, Gwl may be initially activated in the nucleus prior to its nuclear export during the early stage of mitotic entry. The latter possibility may be attributed to the nuclear localization of its activating signal, such as MPF activity, or a lower level of inhibitory activity in the nucleus, such as phosphatases that target Gwl. Finally, Gwl negatively regulates the DNA damage checkpoint in Xenopus egg extracts. It is thus possible that the nuclear localization of Gwl contributes to mitotic entry by suppressing an inhibitory signal, such as the cell cycle checkpoint governing DNA damage or incomplete DNA replication (Yamamoto, 2014).

Drosophila melanogaster importin alpha1 and alpha3 can replace importin alpha2 during spermatogenesis but not oogenesis.

Importin alpha's mediate the nuclear transport of many classical nuclear localization signal (cNLS)-containing proteins. Multicellular animals contain multiple importin alpha genes, most of which fall into three conventional phylogenetic clades, designated alpha1, alpha2, and alpha3. Using degenerate PCR, Drosophila importin alpha1, alpha2, and alpha3 genes were cloned, demonstrating that the complete conventional importin alpha gene family arose prior to the split between invertebrates and vertebrates. The genetic interactions among conventional importin alpha genes were analyzed by studying their capacity to rescue the male and female sterility of importin alpha2 null flies. The sterility of alpha2 null males was rescued to similar extents by importin alpha1, alpha2, and alpha3 transgenes, suggesting that all three conventional importin alpha's are capable of performing the important role of importin alpha2 during spermatogenesis. In contrast, sterility of alpha2 null females was rescued only by importin alpha2 transgenes, suggesting that it plays a paralog-specific role in oogenesis. Female infertility was also rescued by a mutant importin alpha2 transgene lacking a site that is normally phosphorylated in ovaries. These rescue experiments suggest that male and female gametogenesis have distinct requirements for importin alpha2 (Mason, 2002).

Patterns of importin alpha expression during Drosophila spermatogenesis

Importin-alpha proteins do not only mediate the nuclear import of karyophilic proteins but also regulate spindle assembly during mitosis and the assembly of ring canals during Drosophila oogenesis. Three importin-alpha genes are present in the genome of Drosophila. To gain further insights into their function their expression during spermatogenesis was analyzed by using antibodies raised against each of the three Importin-alpha proteins identified in Drosophila, namely, Imp-alpha1, Imp-alpha2, and Imp-alpha3. Each Imp-alpha was found to be expressed during a specific and limited period of spermatogenesis. Strong expression of Imp-alpha2 takes place in spermatogonial cells, persists in spermatocytes, and lasts up to the completion of meiosis. In growing spermatocytes, the intracellular localisation of Imp-alpha2 appears to be dependent upon the rate of cell growth. In pupal testes Imp-alpha2 is essentially present in the spermatocyte nucleus but is localised in the cytoplasm of spermatocytes from adult testes. Both Imp-alpha1 and -alpha3 expression initiates at the beginning of meiosis and ends during spermatid differentiation. Imp-alpha1 expression extends up to the onset of the elongation phase, whereas that of Imp-alpha3 persists up to the completion of nuclear condensation when the spermatids become individualised. During meiosis Imp-alpha1 and -alpha3 are dispersed in the karyoplasm where they are partially associated with the nuclear spindle, albeit not with the asters. At telophase they aggregate around the chromatin. During sperm head differentiation, both Imp-alpha1 and -alpha3 are nuclear. These data indicate that each Imp-alpha protein carries during Drosophila spermatogenesis distinct, albeit overlapping, functions that may involve nuclear import of proteins, microtubule organisation, and other yet unknown processes (Giarre, 2002).


Search PubMed for articles about Drosophila Importin alpha1

Geles, K. G. and Adam, S. A. (2001). Germline and developmental roles of the nuclear transport factor importin alpha3 in C. elegans. Development 128: 1817-1830. PubMed ID: 11311162

Geles, K. G., Johnson, J. J., Jong S. and Adam, S. A. (2002). A role for Caenorhabditis elegans importin IMA-2 in germ line and embryonic mitosis. Mol. Biol. Cell 13: 3138-3147. PubMed ID: 12221121

Giarre, M., et al. (2002). Patterns of importin alpha expression during Drosophila spermatogenesis. J. Struct. Biol. 140 279-290. PubMed ID: 12490175

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Biological Overview

date revised: 15 April 2020

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