Characterization of A 54-kD protein of the inner nuclear membrane: evidence for cell cycle-dependent interaction with the nuclear lamina.

Using a mAb (R-7), we have characterized a 54-kD protein of the chicken nuclear envelope. Based on its biochemical properties and subnuclear distribution p54 is likely to be an integral membrane component specific to the inner nuclear membrane. Fractionation experiments indicate that p54 interacts, directly or indirectly, with the nuclear lamina, and analysis of p54 in cultured cells suggests that this interaction is controlled by cell cycle-dependent posttranslational modification, most likely phosphorylation. Modification of p54 results in a slightly reduced electrophoretic mobility, and it converts the protein from a detergent-resistant to a detergent-extractable form. Detergent solubilization of p54 can be induced in vivo by treating isolated nuclei or nuclear envelopes with highly purified cdc2 kinase, one of the most prominent kinases active in mitotic cells. These results suggest that mitotic phosphorylation of p54 might contribute to control nuclear envelope dynamics during mitosis in vivo.

The Nsp1p carboxy-terminal domain is organized into functionally distinct coiled-coil regions required for assembly of nucleoporin subcomplexes and nucleocytoplasmic transport.

Nucleoporin Nsp1p, which has four predicted coiled-coil regions (coils 1 to 4) in the essential carboxy-terminal domain, is unique in that it is part of two distinct nuclear pore complex (NPC) subcomplexes, Nsp1p-Nup57p-Nup49p-Nic96p and Nsp1p-Nup82p-Nup159p. As shown by in vitro reconstitution, coiled-coil region 2 (residues 673 to 738) is sufficient to form heterotrimeric core complexes and can bind either Nup57p or Nup82p. Accordingly, interaction of Nup82p with Nsp1p coil 2 is competed by excess Nup57p. Strikingly, coil 3 and 4 mutants are still assembled into the core Nsp1p-Nup57p-Nup49p complex but no longer associate with Nic96p. Consistently, the Nsp1p-Nup57p-Nup49p core complex dissociates from the nuclear pores in nsp1 coil 3 and 4 mutant cells, and as a consequence, defects in nuclear protein import are observed. Finally, the nsp1-L640S temperature-sensitive mutation, which maps in coil 1, leads to a strong nuclear mRNA export defect. Thus, distinct coiled-coil regions within Nsp1p-C have separate functions that are related to the assembly of different NPC subcomplexes, nucleocytoplasmic transport, and incorporation into the nuclear pores.

Cloning and sequencing of cDNA clones encoding chicken lamins A and B1 and comparison of the primary structures of vertebrate A- and B-type lamins.

Nuclear lamins are intermediate-filament-type proteins forming a fibrillar meshwork underlying the inner nuclear membrane. The existence of multiple isoforms of lamin proteins in vertebrates is believed to reflect functional specializations during cell division and differentiation. Although biochemical criteria may be used to classify many lamin isoforms into A- and B-type subfamilies, the structural features distinguishing the members of these subfamilies remain to be characterized fully. Here, we report the complete primary structures of chicken lamins A and B1, as they are deduced from cloned cDNAs; in the accompanying paper we present the complete sequence of lamin B2, a second avian B-type lamin. Comparisons of the chicken lamin sequences with each other and with those of other lamins allow us to establish structural features that are common to members of both subfamilies. Conversely, multiple sequence alignments make it possible to identify a number of structural motifs that clearly differentiate B-type lamins from A-type lamins. With this information at hand, we attempt to correlate different biochemical properties of A- and B-type lamins with the presence or absence of specific sequence motifs.

A sensitive homologous radioimmunoassay for human relaxin-2 (h-RLX-2) based on antibodies characterized by epitope mapping studies.

We present a sensitive homologous radioimmunoassay (RIA) for the quantitative determination of human relaxin (hRLX) in human serum, plasma, seminal plasma, and urine. This assay is based on a rabbit antiserum which was generated using recombinant hRLX-2 as immunogen. Using 125I-hRLX-2 as tracer and a total incubation time of 20 - 24 hours the radioimmunoassay showed linearity in a range of 60 - 4000 ng/l, a lower detection limit of 38 ng/l and a mean recovery rate of 98.5%. Intraassay variation was 4.0% (mean = 526 ng/l) and 11.9% (mean = 2368 ng/l), and interassay variation 10.7% (mean = 256 ng/l) and 13.1% (mean = 2368 ng/l). Using hRLX-2 hexapeptides on polystyrene pins, epitopes recognized by the hRLX-2 specific rabbit antiserum were determined experimentally, and compared to predicted epitopes. Both methods led to comparable results. The antiserum, recognizing different epitopes, showed no cross-reactivity with human insulin, hZn-insulin, hIGF-I, hIGF-II, human inhibin alpha-subunit, two different forms of seminal plasma inhibin like peptide, spermolaxin, ubiquitin, prolactin, LH, FSH and hCG.

Connecting viral with cellular interactomes.

Genome-scale screens for intraviral and virus-host protein interactions and the analysis of literature-curated datasets are able to provide a novel, comprehensive perspective of viruses, and virus-infected cells. Until now, large-scale interaction screens were predominantly performed with the yeast-two-hybrid (Y2H) system; however, alternative high-throughput technologies detecting binary protein interactions or protein complexes have been developed. Although many of the previous studies suffer from a rather poor validation of the results and few biological implications, these technologies potentially lead to a plethora of novel hypotheses. Here, we will give an overview of current approaches and their technical limitations, present recent examples and novel developments.

Characterization of nuclear pore protein p62 produced using baculovirus.

Nuclear pore glycoproteins are essential components of the nuclear import apparatus in eukaryotes. In vertebrates, the most abundant of these glycoproteins is a molecule called p62. Like other O-linked N-acetylglucosamine glycoproteins, p62 is normally modified in the cytoplasm and cannot be overexpressed and conveniently collected in a secreted form. We devised an efficient scheme for expression and purification of recombinant p62 from Sf9 cells that may have general applicability for this class of glycoproteins. The purified rat p62 bound to wheat germ agglutinin, consistent with modification by O-linked N-acetylglucosamine. Carbohydrate analysis, in conjunction with amino acid analysis, revealed that baculovirus-expressed rat p62 contains 5-6 mol of N-acetylglucosamine/mol of p62. As observed by circular dichroism, purified p62 expressed in the baculovirus system or in Escherichia coli share essentially the same secondary structure. Purified glycosylated rat p62 will be critical in determining the role of N-acetylglucosamine in both nuclear transport and assembly of the nuclear pore complex.

Role of different domains in the self-association of rat nucleoporin p62.

We have expressed rat nucleoporin p62 cDNA in Escherichia coli to obtain material for structural and self-association studies. Electron microscopy and circular dichroism spectroscopy are consistent with a rod-shaped molecule with an alpha-helical coiled-coil domain at its C terminus and a cross-beta structure at its N terminus, separated by a threonine-rich linker, which has a less-defined secondary structure. Electron microscopy and the solubility properties of fragments produced using thrombin and CNBr digestion indicate that p62 molecules associate to form linear chains and that a small region near the C terminus is an important determinant of assembly. This association may have important consequences for pore structure and function; for example, one way p62 could associate would be to form rings in nuclear pores that could function like barrel hoops.

Nup116p and nup100p are interchangeable through a conserved motif which constitutes a docking site for the mRNA transport factor gle2p.

Nup116p and Nup100p are highly related yeast GLFG nucleoporins, but only Nup116p is stoichiometrically bound to Gle2p, a previously identified mRNA export factor. A short Gle2p-binding sequence within Nup116p (GLEBS; residues 110-166) is sufficient and necessary to anchor Gle2p at the nuclear pores, whereas the carboxy-terminal domain of Nup116p mediates its own nuclear pore complex (NPC) association. The GLEBS is evolutionarily conserved and found in rat/Xenopus Nup98 and an uncharacterized Caenorhabditis elegans ORF, but is absent from Nup100p. When the GLEBS is deleted from Nup116p, Gle2p dissociates from the nuclear envelope and clusters of herniated nuclear pores form. When the GLEBS is inserted into Nup100p, Nup100p-GLEBS complements both the thermosensitive and NPC-herniated phenotype of nup116- cells, and Gle2p is retargeted concomitantly to the NPCs. Thus, the in vivo function of Gle2p is strictly coupled to the short GLEBS within Nup116p which links this putative mRNA transport factor to the nuclear pores.

Nup116p associates with the Nup82p-Nsp1p-Nup159p nucleoporin complex.

Nup116p is a GLFG nucleoporin involved in RNA export processes. We show here that Nup116p physically interacts with the Nup82p-Nsp1p-Nup159p nuclear pore subcomplex, which plays a central role in nuclear mRNA export. For this association, a sequence within the C-terminal domain of Nup116p that includes the conserved nucleoporin RNA-binding motif was sufficient and necessary. Consistent with this biochemical interaction, protein A-Nup116p and the protein A-tagged Nup116p C-terminal domain, like the members of the Nup82p complex, localized to the cytoplasmic side of the nuclear pore complex, as revealed by immunogold labeling. Finally, synthetic lethal interactions were found between mutant alleles of NUP116 and all members of the Nup82p complex. Thus, Nup116p consists of three independent functional domains: 1) the C-terminal part interacts with the Nup82p complex; 2) the Gle2p-binding sequence interacts with Gle2p/Rae1p; and 3) the GLFG domain interacts with shuttling transport receptors such as karyopherin-beta family members.