Nap1 and Chz1 have Separate Htz1 Nuclear Import and Assembly Functions

We analyzed the nuclear import and regulation of the yeast histone variant Htz1 (H2A.Z), and the role of histone chaperones Nap1 and Chz1 in this process. Copurification suggested that Htz1 and H2B dimerized in the cytoplasm prior to import. Like H2B, Htz1 contained a nuclear localization signal (NLS) in its N‐terminus that is recognized by multiple karyopherins (also called importins), indicating multiple transport pathways into the nucleus. However, Kap114 and Kap123 appeared to play the major role in Htz1 import. We also identified a role for Nap1 in the import of Htz1/H2B heterodimers, and Nap1 formed a RanGTP‐insensitive import complex with Htz1/H2B and Kap114. Nap1 was necessary for maintaining a soluble pool of Htz1, indicating that its chaperone function may be important for the dynamic exchange of histones within nucleosomes. In contrast, Chz1 was imported by a distinct import pathway, and Chz1 did not appear to interact with Htz1 in the cytoplasm. Genetic analysis indicated that NAP1 has a function in the absence of HTZ1 that is not shared with CHZ1. This provides further evidence that the histone chaperones Nap1 and Chz1 have separate Htz1‐dependent and ‐independent functions.     

Identification of RanBP2- and kinesin-mediated transport pathways with restricted neuronal and subcellular localization

Ran-binding proteins, karyopherins, and RanGTPase mediate and impart directionality to nucleocytoplasmic transport processes. This biological process remains elusive in neurons. RanBP2 has been localized at the nuclear pore complexes and is very abundant in the neuroretina. RanBP2 mediates the assembly of a large complex comprising RanGTPase, CRM1/exportin-1, importin-β, KIF5-motor proteins, components of the 19S cap of the 26S proteasome, ubc9 and opsin. Here, we show RanBP2 is abundant in the ellipsoid compartment of photoreceptors and RanGTPase-positive particles in cytoplasmic tracks extending away from the nuclear envelope of subpopulations of ganglion cells, suggesting RanBP2's release from nuclear pore complexes. KIF5C and KIF5B are specifically expressed in a subset of neuroretinal cells and differentially localize with RanBP2 and importin-β in distinct compartments. The C-terminal domains of KIF5B and KIF5C, but not KIF5A, associate directly with importin-β in a RanGTPase-dependent fashion in vivo and in vitro, indicating importin-β is an endogenous cargo for a subset of KIF5s in retinal neurons. The KIF5 transport pathway is absent from the myoid region of a topographically distinct subclass of blue cones and the distribution of kinesin-light chains is largely distinct from its KIF5 partners. Altogether, the results identify the existence of neuronal- and subtype-specific kinesin-mediated transport pathways of importin-β-bound cargoes to and/or from RanBP2 and indicate RanBP2 itself may also constitute a scaffold carrier for some of its associated partners. The implications of these findings in protein kinesis and pathogenesis of degenerative neuropathies are discussed.     

Mechanisms of receptor-mediated nuclear import and nuclear export

Nuclear transport of proteins and RNA occurs through the nuclear pore complex and is mediated by a superfamily of transport receptors known collectively as karyopherins. Karyopherins bind to their cargoes by recognition of specific nuclear localization signals or nuclear export signals. Transport through the nuclear pore complex is facilitated by transient interactions between the karyopherins and the nuclear pore complex. The interactions of karyopherins with their cargoes are regulated by the Ras-related GTPase Ran. Ran is assisted in this process by proteins that regulate its GTPase cycle and subcellular localization. In this review, we describe several of the major transport pathways that are conserved in higher and lower eukaryotes, with particular emphasis on the role of Ran. We highlight the latest advances in the structure and function of transport receptors and discuss recent examples of steroid hormone receptor import and regulation by signal transduction pathways. Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity.     

The karyopherin Kap142p/Msn5p mediates nuclear import and nuclear export of different cargo proteins

We have identified a novel pathway for protein import into the nucleus. Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p). We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA replication, DNA repair, and recombination. In wild-type cells, RPA was localized primarily to the nucleus but, in a KAP142 deletion strain, RPA was mislocalized to the cytoplasm and the strain was highly sensitive to bleomycin (BLM). BLM causes DNA double-strand breaks and, in S. cerevisiae, the DNA damage is repaired predominantly by RPA-dependent homologous recombination. Therefore, our results indicate that in wild-type cells a critical portion of RPA was imported into the nucleus by Kap142p. Like several other import-related Kap-substrate complexes, the endogenous RPA-Kap142p complex was dissociated by RanGTP, but not by RanGDP. All three RPA genes are essential for viability, whereas KAP142 is not. Perhaps explaining this disparity, we observed an interaction between RPA and Kap95p in a strain lacking Kap142p. This interaction could provide a mechanism for import of RPA into the nucleus and cell viability in the absence of Kap142p. Together with published results (Kaffman, A., N.M. Rank, E.M. O'Neill, L.S. Huang, and E.K. O'Shea. 1998. Nature. 396:482-486; Blondel, M., P.M. Alepuz, L.S. Huang, S. Shaham, G. Ammerer, and M. Peter. 1999. Genes Dev. 13:2284-2300; DeVit, M.J., and M. Johnston. 1999. Curr. Biol. 9:1231-1241; Mahanty, S.K., Y. Wang, F.W. Farley, and E.A. Elion. 1999. Cell. 98:501-512) our data indicate that the karyopherin Kap142p is able to mediate nuclear import of one set of proteins and nuclear export of a different set of proteins.     

Distinct roles for classical nuclear import receptors in the growth of multinucleated muscle cells

Proper muscle function is dependent on spatial and temporal control of gene expression in myofibers. Myofibers are multinucleated cells that are formed, repaired and maintained by the process of myogenesis in which progenitor myoblasts proliferate, differentiate and fuse. Gene expression is dependent upon proteins that require facilitated nuclear import, however little is known about the regulation of nucleocytoplasmic transport during the formation of myofibers. We analyzed the role of karyopherin alpha (KPNA), a key classical nuclear import receptor, during myogenesis. We established that five karyopherin alpha paralogs are expressed by primary mouse myoblasts in vitro and that their steady-state levels increase in multinucleated myotubes, suggesting a global increase in demand for classical nuclear import during myogenesis. We used siRNA-mediated knockdown to identify paralog-specific roles for KPNA1 and KPNA2 during myogenesis. KPNA1 knockdown increased myoblast proliferation, whereas KPNA2 knockdown decreased proliferation. In contrast, no proliferation defect was observed with KPNA4 knockdown. Only knockdown of KPNA2 decreased myotube growth. These results identify distinct pathways involved in myoblast proliferation and myotube growth that rely on specific nuclear import receptors suggesting that regulation of classical nuclear import pathways likely plays a critical role in controlling gene expression in skeletal muscle.     

Ran-dependent nuclear export mediators: a structural perspective

Nuclear export is an essential eukaryotic activity. It proceeds through nuclear pore complexes (NPCs) and is mediated by soluble receptors that shuttle between nucleus and cytoplasm. RanGTPase-dependent export mediators (exportins) constitute the largest class of these carriers and are functionally highly versatile. All of these exportins load their substrates in response to RanGTP binding in the nucleus and traverse NPCs as ternary RanGTP-exportin-cargo complexes to the cytoplasm, where GTP hydrolysis leads to export complex disassembly. The different exportins vary greatly in their substrate range. Recent structural studies of both protein- and RNA-specific exporters have illuminated how exportins bind their cargoes, how Ran triggers cargo loading and how export complexes are disassembled in the cytoplasm. Here, we review the current state of knowledge and highlight emerging principles as well as prevailing questions.