In eukaryotes, tRNAs are synthesized in the nucleus and after several maturation steps exported to the cytoplasm. Here, we identify exportin-t as a specific mediator of tRNA export. It is a RanGTP-binding, importin beta-related factor with predominantly nuclear localization. It shuttles rapidly between nucleus and cytoplasm and interacts with nuclear pore complexes. Exportin-t binds tRNA directly and with high affinity. Its cellular concentration in Xenopus oocytes was found to be rate-limiting for export of all tRNAs tested, as judged by microinjection experiments. RanGTP regulates the substrate-exportin-t interaction such that tRNA can be preferentially bound in the nucleus and released in the cytoplasm.
-M.Mingot contributed equally to this work Many nuclear transport pathways are mediated by importin b-related transport receptors. Here, we identify human importin (Imp) 4b as well as mouse Imp4a, Imp9a and Imp9b as novel family members. Imp4a mediates import of the ribosomal protein (rp) S3a, while Imp9a and Imp9b import rpS7, rpL18a and apparently numerous other substrates. Ribosomal proteins, histones and many other nuclear import substrates are very basic proteins that aggregate easily with cytoplasmic polyanions such as RNA. Imp9 effectively prevents such precipitation of, for example, rpS7 and rpL18a by covering their basic domains. The same applies to Imp4, Imp5, Imp7 and Impb and their respective basic import substrates. The Impb±Imp7 heterodimer appears specialized for the most basic proteins, such as rpL4, rpL6 and histone H1, and is necessary and suf®cient to keep them soluble in a cytoplasmic environment prior to rRNA or DNA binding, respectively. Thus, just as heat shock proteins function as chaperones for exposed hydrophobic patches, importins act as chaperones for exposed basic domains, and we suggest that this represents a major and general cellular function of importins. Keywords: chaperone/histone/importin/ribosomal protein IntroductionThe nuclear envelope (NE) divides eukaryotic cells into a nuclear and a cytoplasmic compartment, uncouples transcription from translation and thereby necessitates nucleocytoplasmic transport (reviewed in Mattaj and Englmeier, 1998;Nakielny and Dreyfuss, 1999). tRNAs, mRNAs and rRNAs, for example, need to be exported from the nucleus to the cytoplasm, where they mediate translation. Conversely, virtually all nuclear functions are dependent on proteins which are imported from the cytoplasm. The nuclear compartment already represents the ®nal destination for many import substrates, such as histones or polymerases. Numerous other proteins, however, pass through nuclei only transiently. Ribosomal proteins, for example, are ®rst imported, assemble in the nucleoli with rRNAs, and ®nally become exported as ribosomal subunits to the cytoplasm (for a review see Venema and Tollervey, 1999).Macromolecular transport between the nucleus and cytoplasm proceeds through nuclear pore complexes (NPCs) and is normally receptor mediated. Impb-type transport receptors account for most, but not all, nuclear transport pathways (for reviews see Mattaj and Englmeier, 1998;Nakielny and Dreyfuss, 1999;Conti and Izaurralde, 2001). They constitute a diverse protein superfamily and occur in two forms, import mediators (importins) and exportins. They circulate between nucleus and cytoplasm, recognize cargo molecules and transfer them from one side of the NE to the other. Substrate loading and release is guided by a gradient of RanGTP across the NE, whereby a high nuclear RanGTP concentration favours cargo loading onto exportins and substrate displacement from importins, while cytoplasmic conditions with low RanGTP levels release substrates from exportins but allow importin±cargo complexes to fo...
Immunizations using the endoplasmic reticulum-resident heat shock protein Gp96 induce specific immune responses. Specificity is based on the major histocompatibility complex class I-restricted crosspresentation of Gp96-associated peptides derived from endogenous proteins. Initiation of the immune response depends on the ability of Gp96 to induce the production of proinflammatory cytokines by macrophages and dendritic cells (DCs) and of their maturation in a fashion presumably independent of associated peptide. Both events are mediated by Gp96 receptors on antigen-presenting cells. It is known that Gp96 is released from cells at necrosis induced, for example, by virus infection. Although this event supports the efficient induction of immune responses, it might also interfere with processes that are susceptible to chronic inflammation, such as wound healing after tissue damage. Therefore, Gp96-mediated stimulation of the immune system requires tight regulation. Here we show that human thrombocytes specifically interact with Gp96 and that binding of Gp96 to platelets is enhanced more than 10-fold on activation by thrombin. Gp96 interferes with neither thrombin-induced platelet activation nor platelet aggregation. However, the presence of platelets during Gp96-mediated DC activation reduces the secretion of proinflammatory cytokines and the activation of DCs. This effect is independent of soluble platelet factors and cell-to-cell contact between DCs and thrombocytes. Thus, we provide evidence for a regulatory mechanism that neutralizes Gp96 molecules systemically, especially in the blood. This effect might be of significance in wounds in which chronic inflammation and immune responses against autoantigens have to be prevented. IntroductionThe endoplasmic reticulum (ER)-resident heat shock protein (HSP) Gp96 plays multiple roles in mammalian organisms. As a chaperone, it assists protein folding and prevents aggregation of partially unfolded proteins in the ER. 1 In this key compartment of the major histocompatibility (MHC) class I presentation pathway, Gp96 is also one of the major peptide-binding proteins, and it associates with a peptide pool representative for the protein content of the cell. 2,3 Gp96 is released from cells after tissue damage caused by severe injury and resulting from necrotic cell death induced by freeze-thaw cycles 4 or virus infection. 5 Its presence in the extracellular space reveals surprising immunostimulatory properties: immunization with Gp96 preparations from tumor cells has been shown to elicit protective and therapeutic immune responses against the tumor from which the HSP had been purified. 6,7 The specificity of this immune response is attributed to tumor-derived peptides associated with Gp96. 6 After receptor-mediated endocytosis of Gp96 by professional antigen-presenting cells (APCs), 8 these peptides are presented on MHC class I molecules. 9 This process is usually referred to as cross-presentation, 10,11 and it is one of the key events during the priming of naive T cells. 12 In ...
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