Susanne Kostka

Export of Importin α from the Nucleus Is Mediated by a Specific Nuclear Transport Factor

Abstract NLS proteins are transported into the nucleus by the importin α/β heterodimer. Importin α binds the NLS, while importin β mediates translocation through the nuclear pore complex. After translocation, RanGTP, whose predicted concentration is high in the nucleus and low in the cytoplasm, binds importin β and displaces importin α. Importin α must then be returned to the cytoplasm, leaving the NLS protein behind. Here, we report that the previously identified CAS protein mediates importin α re-export. CAS binds strongly to importin α only in the presence of RanGTP, forming an importin α/CAS/RanGTP complex. Importin α is released from this complex in the cytoplasm by the combined action of RanBP1 and RanGAP1. CAS binds preferentially to NLS-free importin α, explaining why import substrates stay in the nucleus.

Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope

BACKGROUND Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60kD importin protein (importin 60) as a single polypeptide. RESULTS We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. CONCLUSIONS Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.

Posttranslational protein transport in yeast reconstituted with a purified complex of Sec proteins and Kar2p

We have reproduced the posttranslational mode of protein translocation across the endoplasmic reticulum membrane with reconstituted proteoliposomes containing a purified complex of seven yeast proteins. This Sec complex includes a heterotrimeric Sec61p complex, homologous to that in mammals, as well as all other membrane proteins found in genetic screens for translocation components. Efficient posttranslational translocation also requires the addition of lumenal Kar2p (BiP) and ATP. The trimeric Sec61p complex also exists as a separate entity that, in contrast with the large Sec complex, is associated with membrane-bound ribosomes. We therefore hypothesize that distinct membrane protein complexes function in co- and posttranslational translocation pathways.

Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes.

Transport receptors of the importin β superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co‐operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment‐specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin β family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF‐5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF‐5A appears to be complex and to involve the hypusine modification that is unique to eIF‐5A. We discuss possible cellular roles for nuclear export of eIF‐5A.

Importin 13: a novel mediator of nuclear import and export

Importin β‐related receptors mediate translocation through nuclear pore complexes. Co‐operation with the RanGTPase system allows them to bind and subsequently release their substrates on opposite sides of the nuclear envelope, which in turn ensures a directed nucleocytoplasmic transport. Here we identify a novel family member from higher eukaryotes that functions primarily, but not exclusively, in import. It accounts for nuclear accumulation of the SUMO‐1/sentrin‐conjugating enzyme hUBC9 and mediates import of the RBM8 (Y14) protein, and is therefore referred to as importin 13 (Imp13). Unexpectedly, Imp13 also shows export activity towards the translation initiation factor eIF1A and is thus a case where a single importin β‐like receptor transports different substrates in opposite directions. However, Imp13 operates differently from typical exportins in that the binding of eIF1A to Imp13 is only regulated indirectly by RanGTP, and the cytoplasmic release of eIF1A from Imp13 is triggered by the loading of import substrates onto Imp13.

Importin Provides a Link between Nuclear Protein Import and U snRNA Export

Importin-alpha mediates nuclear protein import by binding nuclear localization signals and importin-beta. We find approximately 30% of SRP1p, the yeast importin-alpha, in a nuclear complex with the Saccharomyces cerevisiae nuclear cap-binding protein complex (CBC). Similarly, a large fraction of Xenopus CBC is associated with importin-alpha in the nucleus. CBC promotes nuclear export of capped U snRNAs and shuttles between nucleus and cytoplasm. The CBC-importin-alpha complex binds specifically to capped RNA, suggesting that CBC might shuttle while bound to importin-alpha. Strikingly, importin-beta binding displaces the RNA from the CBC-importin-alpha complex. Thus, the commitment of CBC for nuclear reentry triggers the release of the export substrate into the cytoplasm. We provide evidence for a mechanism that ensures that importin-mediated RNA release is a specifically cytoplasmic event.

Analysis of mammalian 20S proteasome biogenesis: the maturation of beta-subunits is an ordered two-step mechanism involving autocatalysis.

Maturation of eukaryotic 20S proteasomes involves the processing of beta‐subunits by limited proteolysis. To study the processing mechanism we analysed different point mutations of the beta‐subunit LMP2 in transfected human T2 cells. Here we show that the presence of the intact Gly‐1Thr1 consensus motif and Lys33 are essential for correct processing. Mutation of Thr1, the active site residue in mature subunits, or of Lys33, results in complete inhibition of processing at the consensus site. In addition, proprotein processing in vitro of wild‐type LMP2, incorporated in immature 16S precursor complexes, can be blocked by a proteasome‐specific inhibitor. While the processing of inhibitor‐treated wild‐type proprotein was completely prevented, the site‐directed mutagenesis of LMP2 results in processing intermediates carrying an extension of 8–10 residues preceding Thr1, suggesting an additional cleavage event within the prosequence. Furthermore, exchange of mammalian prosequences interferes with processing efficiency and suggests subunit specificity. Based on our data we propose a model for self‐activation of proteasomal beta‐subunits in which residue Thr1 serves as nucleophile and Lys33 as proton donor/acceptor. We provide evidence that subunit processing of mammalian beta‐subunits proceeds via a novel ordered two‐step mechanism involving autocatalysis.

Sm protein–Sm site RNA interactions within the inner ring of the spliceosomal snRNP core structure

Seven Sm proteins, E, F, G, D1, D2, D3 and B/B′, assemble in a stepwise manner onto the single‐stranded Sm site element (PuAU4–6GPu) of the U1, U2, U4 and U5 spliceosomal snRNAs, resulting in a doughnut‐shaped core RNP structure. Here we show by UV cross‐linking experiments using an Sm site RNA oligonucleotide (AAUUUUUGA) that several Sm proteins contact the Sm site RNA, with the most efficient cross‐links observed for the G and B/B′ proteins. Site‐specific photo‐cross‐linking revealed that the G and B/B′ proteins contact distinct uridines (in the first and third positions, respectively) in a highly position‐specific manner. Amino acids involved in contacting the RNA are located at equivalent regions in both proteins, namely in loop L3 of the Sm1 motif, which has been predicted to jut into the hole of the Sm ring. Our results thus provide the first evidence that, within the core snRNP, multiple Sm protein–Sm site RNA contacts occur on the inner surface of the heptameric Sm protein ring.

Overexpression of the proteasome subunits LMP2, LMP7, and MECL-1, but not PA28 alpha/beta, enhances the presentation of an immunodominant lymphocytic choriomeningitis virus T cell epitope.

The proteasome is a large protease complex that generates most of the peptide ligands of MHC class I molecules either in their final form or in the form of N-terminally extended precursors. Upon the stimulation of cells with IFN-γ, three constitutively expressed subunits of the 20S proteasome are replaced by the inducible subunits LMP2 (low-molecular mass polypeptide 2), LMP7, and MECL-1 (multicatalytic endopeptidase complex-like-1) to form so-called immunoproteasomes. We show in this study that overexpression of these three subunits in triple transfectants led to a marked enhancement in the H-2Ld-restricted presentation of the immunodominant nonameric epitope NP118, which is derived from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. Overexpression of the α and β subunits of the IFN-γ-inducible proteasome regulator PA28, in contrast, did not have a comparable effect. In vitro, immunoproteasomes as compared with constitutive proteasomes generated higher amounts of 11- and 12-mer fragments containing the NP118 epitope. These are likely to be cytosolic precursors of NP118, as a proline anchor residue in the second position of NP118 may interfere with TAP-mediated transport of the nonameric epitope itself. In conclusion, we provide evidence that up-regulation of the three inducible subunits, LMP2, LMP7, and MECL-1, can result in a marked improvement of Ag presentation and that, depending on the epitope, PA28 and immunoproteasomes may differentially affect Ag processing.

The Selective Proteasome Inhibitors Lactacystin and Epoxomicin Can Be Used to Either Up- or Down-Regulate Antigen Presentation at Nontoxic Doses

The complete inhibition of proteasome activities interferes with the production of most MHC class I peptide ligands as well as with cellular proliferation and survival. In this study we have investigated how partial and selective inhibition of the chymotrypsin-like activity of the proteasome by the proteasome inhibitors lactacystin or epoxomicin would affect Ag presentation. At 0.5–1 μM lactacystin, the presentation of the lymphocytic choriomeningitis virus-derived epitopes NP118 and GP33 and the mouse CMV epitope pp89–168 were reduced and were further diminished in a dose-dependent manner with increasing concentrations. Presentation of the lymphocytic choriomeningitis virus-derived epitope GP276, in contrast, was markedly enhanced at low, but abrogated at higher, concentrations of either lactacystin or epoxomicin. The inhibitor-mediated effects were thus epitope specific and did not correlate with the degradation rates of the involved viral proteins. Although neither apoptosis induction nor interference with cellular proliferation was observed at 0.5–1 μM lactacystin in vivo, this concentration was sufficient to alter the fragmentation of polypeptides by the 20S proteasome in vitro. Our results indicate that partial and selective inhibition of proteasome activity in vivo is a valid approach to modulate Ag presentation, with potential applications for the treatment of autoimmune diseases and the prevention of transplant rejection.