Crystal structure of human Karyopherin β2 bound to the PY-NLS of Saccharomyces cerevisiae Nab2

Import-Karyopherin or Importin proteins bind nuclear localization signals (NLSs) to mediate the import of proteins into the cell nucleus. Karyopherin β2 or Kapβ2, also known as Transportin, is a member of this transporter family responsible for the import of numerous RNA binding proteins. Kapβ2 recognizes a targeting signal termed the PY-NLS that lies within its cargos to target them through the nuclear pore complex. The recognition of PY-NLS by Kapβ2 is conserved throughout eukaryotes. Kap104, the Kapβ2 homolog in Saccharomyces cerevisiae, recognizes PY-NLSs in cargos Nab2, Hrp1, and Tfg2. We have determined the crystal structure of Kapβ2 bound to the PY-NLS of the mRNA processing protein Nab2 at 3.05-Å resolution. A seven-residue segment of the PY-NLS of Nab2 is observed to bind Kapβ2 in an extended conformation and occupies the same PY-NLS binding site observed in other Kapβ2·PY-NLS structures.     

Efficient nuclear transport of structurally disturbed cargo: mutations in a cargo protein switch its cognate karyopherin

The Karyopherin (Kap) family of nuclear transport receptors enables trafficking of proteins to and from the nucleus in a precise, regulated manner. Individual members function in overlapping pathways, while simultaneously being very specific for their main cargoes. The details of this apparent contradiction and rules governing pathway preference remain to be further elucidated. S. cerevisiae Lhp1 is an abundant protein that functions as an RNA chaperone in a variety of biologically important processes. It localizes almost exclusively to the nucleus and is imported by Kap108. We show that mutation of 3 of the 275 residues in Lhp1 alters its import pathway to a Kap121-dependent process. This mutant does not retain wild-type function and is bound by several chaperones. We propose that Kap121 also acts as a chaperone, one that can act as a genetic buffer by transporting mutated proteins to the nucleus.     

Impaired nuclear transport induced by juvenile ALS causing P525L mutation in NLS domain of FUS: A molecular mechanistic study

Amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD) are progressive neurological disorders affecting motor neurons. Cellular aggregates of fused in sarcoma (FUS) protein are found in cytoplasm of ALS and FTLD patients. Nuclear localisation signal (NLS) domain of FUS binds to Karyopherin β2 (Kapβ2), which drives nuclear transport of FUS from cytoplasm. Several pathogenic mutations are reported in FUS NLS, which are associated with its impaired nuclear transport and cytoplasmic mis-localisation. P525L mutation in NLS is most commonly found in cases of juvenile ALS (jALS), which affects individuals below 25 years of age. jALS progresses aggressively causing death within a year of its onset. This study elucidates the molecular mechanism behind jALS-causing P525L mutation hindering nuclear transport of FUS. We perform multiple molecular dynamics simulations in aqueous and hydrophobic solvent to understand the effect of the mutation at molecular level. Dynamics of Kapβ2-FUS complex is better captured in hydrophobic solvent compared to aqueous solvent. P525 and Y526 (PY-motif) of NLS exhibit fine-tuned stereochemical arrangement, which is essential for optimum Kapβ2 binding. P525L causes loss of several native contacts at interface leading to weaker binding, which promotes self-aggregation of FUS in cytoplasm. Native complex samples closed conformation, while mutant complex exhibits open conformation exposing hydrophilic residues of Kapβ2 to hydrophobic solvent. Mutant complex also fails to exhibit spring-like motion essential for its transport through nuclear pore complex. This study provides a mechanistic insight of binding affinity between NLS and Kapβ2 that inhibits self-aggregation of FUS preventing the disease condition.     

Mutational analysis of H3 and H4 N termini reveals distinct roles in nuclear import

Core histones H3 and H4 are rapidly imported into the nucleus by members of the karyopherin (Kap)/importin family. We showed that H3 and H4 interact with Kap123p, histone acetyltransferase-B complex (HAT-B), and Asf1p in cytosol. In vivo analysis indicated that Kap123p is required for H3-mediated import, whereas H4 utilizes multiple Kaps including Kap123p. The evolutionary conservation of H3 and H4 cytoplasmic acetylation led us to analyze the role of acetylation in nuclear transport. We determined that lysine 14 is critical for H3 NLS function in vivo and demonstrated that mutation of H3 lysine 14 to the acetylation-mimic glutamine decreased association with Kap123p in vitro. Several lysines in the H4 NLS are important for its function. We showed that mutation of key lysines to glutamine resulted in a greater import defect than mutation to arginine, suggesting that positive charge promotes NLS function. Lastly we determined that six of ten N-terminal acetylation sites in H3 and H4 can be mutated to arginine, indicating that deposition acetylation is not absolutely necessary in vivo. However, the growth defect of these mutants suggests that acetylation does play an important role in import. These findings suggest a model where cytosolic histones bind import karyopherins prior to acetylation. Other factors are recruited to this complex such as HAT-B and Asf1p; these factors in turn promote acetylation. Acetylation may be important for modulating the interaction with transport factors and may play a role in the release of histones from karyopherins in the nucleus.     

Karyopherins in nuclear pore biogenesis: a role for Kap121p in the assembly of Nup53p into nuclear pore complexes

The mechanisms that govern the assembly of nuclear pore complexes (NPCs) remain largely unknown. Here, we have established a role for karyopherins in this process. We show that the yeast karyopherin Kap121p functions in the targeting and assembly of the nucleoporin Nup53p into NPCs by recognizing a nuclear localization signal (NLS) in Nup53p. This karyopherin-mediated function can also be performed by the Kap95p-Kap60p complex if the Kap121p-binding domain of Nup53p is replaced by a classical NLS, suggesting a more general role for karyopherins in NPC assembly. At the NPC, neighboring nucleoporins bind to two regions in Nup53p. One nucleoporin, Nup170p, associates with a region of Nup53p that overlaps with the Kap121p binding site and we show that they compete for binding to Nup53p. We propose that once targeted to the NPC, dissociation of the Kap121p-Nup53p complex is driven by the interaction of Nup53p with Nup170p. At the NPC, Nup53p exists in two separate complexes, one of which is capable of interacting with Kap121p and another that is bound to Nup170p. We propose that fluctuations between these two states drive the binding and release of Kap121p from Nup53p, thus facilitating Kap121p's movement through the NPC.