SummaryDuring interphase, the nuclear envelope (NE) serves as a selective barrier between cytosol and nucleoplasm. When vertebrate cells enter mitosis, the NE is dismantled in the process of nuclear envelope breakdown (NEBD). Disassembly of nuclear pore complexes (NPCs) is a key aspect of NEBD, required for NE permeabilization and formation of a cytoplasmic mitotic spindle. Here, we show that both CDK1 and polo-like kinase 1 (PLK1) support mitotic NPC disintegration by hyperphosphorylation of Nup98, the gatekeeper nucleoporin, and Nup53, a central nucleoporin linking the inner NPC scaffold to the pore membrane. Multisite phosphorylation of Nup53 critically contributes to its liberation from its partner nucleoporins, including the pore membrane protein NDC1. Initial steps of NPC disassembly in semi-permeabilized cells can be reconstituted by a cocktail of mitotic kinases including cyclinB-CDK1, NIMA, and PLK1, suggesting that the unzipping of nucleoporin interactions by protein phosphorylation is an important principle underlying mitotic NE permeabilization.

Immunoproteasomes are considered to be optimised to process Ags and to alter the peptide repertoire by generating a qualitatively different set of MHC class I epitopes. Whether the immunoproteasome at the biochemical level, influence the quality rather than the quantity of the immuno-genic peptide pool is still unclear. Here, we quantified the cleavage-site usage by human standard-and immunoproteasomes, and proteasomes from immuno-subunit-deficient mice, as well as the peptides generated from model polypeptides. We show in this study that the different proteasome isoforms can exert significant quantitative differences in the cleavage-site usage and MHC class I restricted epitope production. However, independent of the proteasome isoform and substrates studied, no evidence was obtained for the abolishment of the specific cleavage-site usage, or for differences in the quality of the peptides generated. Thus, we conclude that the observed differences in MHC class I restricted Ag presentation between standard-and immunoproteasomes are due to quantitative differences in the proteasome-generated antigenic peptides.Keywords: Antigen presentation r Immunoproteasome r MHC class I restricted epitopes r Proteasome r Proteolysis See accompanying Commentary by Zanker and ChenAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe 20S proteasome is the central proteolytic machinery of the ubiquitin proteasome system, being responsible for the main Correspondence: Dr. Michele Mishto e-mail: michele.mishto@charite.de part of extra-lysosomal protein degradation and generation of MHC class I restricted epitopes [1]. During evolution, the 20S proteasome retained a conserved structure of four stacked seven membered rings (α 7 β 7 β 7 α 7 ). In each β ring, the 20S standard proteasome has three catalytic standard subunits (i.e. β1s, β2s and β5s) that carry an N-terminal threonine residue as a reactive nucleophile. Based on the analysis of yeast 20S proteasome active site mutants with short fluorogenic peptide substrates C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2014. 44: 3508-3521 Antigen processing 3509 chymotryptic-, tryptic-and caspase-like activities were assigned to the β5, β2 and β1 subunits, respectively [2]. Larger polypeptide substrates bind with their residues surrounding the cleavage site, that is residues in position P4 to P1 (cleavage site) and P1 to P4 , to the non-primed and primed substrate binding sites [3] of the proteasome, respectively. This provides the stability and the orientation of the substrate, thereby determining the cleavage-site usage within a protein substrate [4]. In mammalia, the cytokine IFN-γ induces the expression of three active sites carrying alternative β1i/LMP2, β2i/MECL1 and β5i/LMP7 immuno-subunits, and in consequence the formation of the immunoproteasome isoforms [5].Since the β1i and β5i immuno-subunits are encoded within the MHC class II region in close neighbourhood to ...

SummaryAlthough nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus proteins pUL31 and pUL34 form the heterodimeric nuclear egress complex (NEC), which, in turn, mediates the formation of tight-fitting membrane vesicles around capsids at the inner nuclear membrane. Here, we present the crystal structure of the pseudorabies virus NEC. The structure revealed that a zinc finger motif in pUL31 and an extensive interaction network between the two proteins stabilize the complex. Comprehensive mutational analyses, characterized both in situ and in vitro, indicated that the interaction network is not redundant but rather complementary. Fitting of the NEC crystal structure into the recently determined cryoEM-derived hexagonal lattice, formed in situ by pUL31 and pUL34, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling.

Short-lived proteins are degraded by proteasome complexes, which contain a proteolytic core particle (CP) but differ in the number of regulatory particles (RPs) and activators. A recently described member of conserved proteasome activators is Blm10. Blm10 contains 32 HEATlike modules and is structurally related to the nuclear import receptor importin/karyopherin b. In proliferating yeast, RP-CP assemblies are primarily nuclear and promote cell division. During quiescence, RP-CP assemblies dissociate and CP and RP are sequestered into motile cytosolic proteasome storage granuli (PSG). Here, we show that CP sequestration into PSG depends on Blm10, whereas RP sequestration into PSG is independent of Blm10. PSG rapidly clear upon the resumption of cell proliferation and proteasomes are relocated into the nucleus. Thereby, Blm10 facilitates nuclear import of CP. Blm10-bound CP serves as an import receptor-cargo complex, as Blm10 mediates the interaction with FG-rich nucleoporins and is dissociated from the CP by Ran-GTP. Thus, Blm10 represents the first CP-dedicated nuclear import receptor in yeast.

The nucleus is enclosed by the nuclear envelope, a double membrane which creates a selective barrier between the cytoplasm and the nuclear interior. Its barrier and transport characteristics are determined by nuclear pore complexes (NPCs) that are embedded within the nuclear envelope, and control molecular exchange between the cytoplasm and nucleoplasm. In this Commentary, we discuss the biogenesis of these huge protein assemblies from approximately one thousand individual proteins. We will summarize current knowledge about distinct assembly modes in animal cells that are characteristic for different cell cycle phases and their regulation.

RECQL4, which is mutated in the Rothmund–Thomson syndrome characterized by premature aging and cancer susceptibility, is a microtubule-associated protein required for mitotic chromosome alignment.

The eukaryotic nucleus remodels extensively during mitosis. Upon mitotic entry, the nuclear envelope breaks down and chromosomes condense into rod-shaped bodies, which are captured by the spindle apparatus and segregated during anaphase. Through telophase, chromosomes decondense and the nuclear envelope reassembles, leading to a functional interphase nucleus. While the molecular processes occurring in early mitosis are intensively investigated, our knowledge about molecular mechanisms of nuclear reassembly is rather limited. Using cell free and cellular assays, we identify the histone variant H2A.Z and its chaperone VPS72/YL1 as important factors for reassembly of a functional nucleus after mitosis. Live-cell imaging shows that siRNA-mediated downregulation of VPS72 extends the telophase in HeLa cells. In vitro, depletion of VPS72 or H2A.Z results in malformed and nonfunctional nuclei. VPS72 is part of two chromatin-remodeling complexes, SRCAP and EP400. Dissecting the mechanism of nuclear reformation using cell-free assays, we, however, show that VPS72 functions outside of the SRCAP and EP400 remodeling complexes to deposit H2A.Z, which in turn is crucial for formation of a functional nucleus.

Lack of high-quality antibodies against transmembrane proteins is a widely recognized hindrance in biomedical and cell biological research. Here we present a robust pipeline for the generation of polyclonal antibodies employing full-length membrane proteins as immunogens to overcome this “antibody bottleneck”. We express transmembrane proteins fused to a MISTIC fragment that enhances expression of eukaryotic membrane proteins in E. coli. Purified membrane proteins are used as immunogen for rabbit injection employing standard immunizing protocols. The raised antibodies against membrane proteins of the endoplasmic reticulum and the nuclear envelope, which we use as test cases, function in a wide range of applications and are superior to ones produced against soluble domains as immunogens.