An integral membrane protein from the nuclear pore complex is also present in the annulate lamellae: implications for annulate lamella formation

Annulate lamellae (AL) are cytoplasmic arrays of stacked membrane cisternae containing densely packed pore complexes which are similar in structure to the nuclear pore complexes (NPCs) and thus referred to as annulate lamella pore complexes (ALPCs). We have recently shown that the integral nuclear pore membrane protein POM121 tagged with green fluorescent protein was correctly targeted to the nuclear pores (H. S?derqvist et al., 1997, Eur. J. Biochem. 250, 808-813). Here we have investigated if POM121 fused to three tandem molecules of yellow fluorescent protein (YFP) (POM121-YFP(3)) also was able to distribute in the extensive and well-characterized AL of RC37 and BMGE cells. Transfected RC37 or BMGE cells displayed YFP fluorescence around the nuclear envelope, as well as in the cytoplasmic AL structures. The YFP fluorescence colocalized perfectly with immunostaining using antibodies specific for different NPC proteins. The AL of both transfected and untransfected BMGE cells resisted extractions with Tx-100 and 250 mM NaCl, but were completely solubilized at 450 mM NaCl. Loss of YFP fluorescence and immunostaining for other NPC proteins correlated under all extraction conditions tested, suggesting that overexpressed POM121-YFP(3) had become an integrated part both of the NPCs and of the ALPCs. Furthermore, we have generated a stable BHK cell line expressing POM121-YFP(3) located exclusively at the nuclear pores. Treatment with vinblastine sulfate, which induces formation of AL in a variety of cells, resulted in distribution of POM121-YFP(3) into cytoplasmic foci colocalizing with immunostaining for peripheral NPC proteins. Taken together, the results show that YFP-tagged POM121 is able to distribute in drug-induced or naturally occurring AL, suggesting that POM121 is a natural constituent of ALPCs. In COS cells, which normally lack or have very little AL, YFP-tagged POM121 distributed in the nuclear pores when expressed at low levels. However, at high expression levels the YFP fluorescence also distributed in a number of brightly fluorescing cytoplasmic dots or foci, which were not present in untransfected cells. This was also true for untagged POM121. The cytoplasmic foci varied in size from 0. 1 to 2 microm and were distinctly located in the immediate vicinity of ER cisternae (without colocalizing) and also contained other nuclear pore proteins, indicating that they may represent cytoplasmic AL. This idea is supported by time-lapse studies of postmitotic assembly of these structures. This raises the question of the role of POM121 in ALPC and NPC biogenesis.     

ER retention may play a role in sorting of the nuclear pore membrane protein POM121

Integral membrane proteins of the nuclear envelope (NE) are synthesized on the rough endoplasmic reticulum (ER) and following free diffusion in the continuous ER/NE membrane system are targeted to their proper destinations due to interactions of specific domains with other components of the NE. By studying the intracellular distribution and dynamics of a deletion mutant of an integral membrane protein of the nuclear pores, POM121, which lacks the pore-targeting domain, we investigated if ER retention plays a role in sorting of integral membrane proteins to the nuclear envelope. A nascent membrane protein lacking sorting determinants is believed to diffuse laterally in the continuous ER/NE lipid bilayer and expected to follow vesicular traffic to the plasma membrane. The GFP-tagged deletion mutant, POM121(1-129)-GFP, specifically distributed within the ER membrane, but was completely absent from the Golgi compartment and the plasma membrane. Experiments using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that despite having very high mobility within the whole ER network (D = 0.41 +/- 0.11 micro m(2)/s) POM121(1-129)-GFP was unable to exit the ER. It was also not detected in post-ER compartments of cells incubated at 15 degrees C. Taken together, these experiments show that amino acids 1-129 of POM121 are able to retain GFP in the ER membrane and suggest that this retention occurs by a direct mechanism rather than by a retrieval mechanism. Our data suggest that ER retention might be important for sorting of POM121 to the nuclear pores.     

POM121 and Sun1 play a role in early steps of interphase NPC assembly

Nuclear pore complexes (NPCs) assemble at the end of mitosis during nuclear envelope (NE) reformation and into an intact NE as cells progress through interphase. Although recent studies have shown that NPC formation occurs by two different molecular mechanisms at two distinct cell cycle stages, little is known about the molecular players that mediate the fusion of the outer and inner nuclear membranes to form pores. In this paper, we provide evidence that the transmembrane nucleoporin (Nup), POM121, but not the Nup107-160 complex, is present at new pore assembly sites at a time that coincides with inner nuclear membrane (INM) and outer nuclear membrane (ONM) fusion. Overexpression of POM121 resulted in juxtaposition of the INM and ONM. Additionally, Sun1, an INM protein that is known to interact with the cytoskeleton, was specifically required for interphase assembly and localized with POM121 at forming pores. We propose a model in which POM121 and Sun1 interact transiently to promote early steps of interphase NPC assembly.     

An integral membrane protein of the pore membrane domain of the nuclear envelope contains a nucleoporin-like region

We have identified an integral membrane protein of 145 kD (estimated by SDS-PAGE) of rat liver nuclear envelopes that binds to WGA. We obtained peptide sequence from purified p145 and cloned and sequenced several cDNA clones and one genomic clone. The relative molecular mass of p145 calculated from its complete, cDNA deduced primary structure is 120.7 kD. Antibodies raised against a synthetic peptide represented in p145 reacted monospecifically with p145. In indirect immunofluorescence these antibodies gave punctate staining of the nuclear envelope. Immunogold EM showed specific decoration of the nuclear pores. Thus p145 is an integral membrane protein located specifically in the "pore membrane" domain of the nuclear envelope. To indicate this specific location, and based on its calculated relative molecular mass, the protein is termed POM 121 (pore membrane protein of 121 kD). The 1,199- residue-long primary structure shows a hydrophobic region (residues 29- 72) that is likely to form one (or two adjacent) transmembrane segment(s). The bulk of the protein (residues 73-1199) is predicted to be exposed not on the cisternal side but on the pore side of the pore membrane. It contains 36 consensus sites for various kinases. However, its most striking feature is a repetitive pentapeptide motif XFXFG that has also been shown to occur in several nucleoporins. This nucleoporin- like domain of POM 121 is proposed to function in anchoring components of the nuclear pore complex to the pore membrane.     

Noninvasive Monitoring of Apoptosis versus Necrosis in a Neuroblastoma Cell Line Expressing a Nuclear Pore Protein Tagged with the Green Fluorescent Protein

A fusion chimera between the integral nuclear pore membrane protein POM121 and GFP (green fluorescent protein) has been shown to correctly target to the nuclear pores when transiently expressed in a number of mammalian cell types. POM121-GFP is therefore an excellent marker for the noninvasive studies of the nuclear pores in living cells using fluorescence microscopy. We have established a line of neuroblastoma cells stably expressing the POM121-GFP fusion protein. We also monitored the nuclear envelope in living cells after induction of apoptosis or necrosis using 1 μM staurosporine or 100 μMp-benzoquinone, respectively. Interestingly, the POM121-GFP fluorescence was weaker or missing in the apoptotic cells. The disappearance of the nuclear pore marker accompanied apoptotic progression as judged by the degree of chromatin condensation and DNA fragmentation as analyzed by DNA staining and TUNEL assay, respectively. In contrast, the intensity of the nuclear rim fluorescence was unaffected in necrotic cells displaying an abnormal morphology with tilted nuclei. Thus, it was possible to distinguish between apoptotic and necrotic development in living cells using fluorescence microscopy. This cell line provides a fast and convenient model for screening suspected toxic xenobiotics.     

A novel gene (PLU-1) containing highly conserved putative DNA/chromatin binding motifs is specifically up-regulated in breast cancer.

A novel human gene (PLU-1) has been identified which shows a highly restricted expression in normal adult tissues but which is consistently expressed in breast cancers. A fragment of the PLU-1 cDNA was identified by differentially screening a fetal brain library with cDNAs prepared from ce-1 cells (a human mammary epithelial cell line overexpressing c-ErbB2) treated or untreated with the antibody 4D5, which inhibits c-ErbB2 phosphorylation. Clones covering the full cDNA sequence of 6.4 kilobases were isolated from a breast cancer cDNA library. Although expression of PLU-1 in ce-1 cells is regulated by signaling from c-ErbB2, the gene is expressed in all the breast cancer cell lines examined, in cells cultured from primary breast cancers, and in the invasive and in situ components of primary breast cancers. Translation of the open reading frame predicts a protein of 1544 amino acids, which contains three PHD/LAP motifs, a specific DNA-binding domain found in a Drosophila protein (dri) and novel domains showing extensive homology with other human and non human gene products. Transient transfection of cell lines with MYC-tagged PLU-1 showed the protein to be localized in the nucleus and associated with discrete foci. The presence of the dri motif and PHD/LAP fingers together with the clear nuclear localization and consistent expression in breast cancers, suggest a role for PLU-1 in regulating gene expression in breast cancers.     

Degradation of GFP-labelled POM121, a non-invasive sensor of nuclear apoptosis, precedes clustering of nuclear pores and externalisation of phosphatidylserine

The nuclear pore membrane protein POM121 is specifically degraded during apoptosis by a caspase-3-dependent process enabling early detection of apoptosis in living cells expressing POM121-GFP. Here we further investigated temporal aspects of apoptotic degradation of POM121-GFP. We demonstrate that decreased POM121-GFP fluorescence precedes annexin V-labelling of apoptotic cells. This indicates that degradation of the nuclear pore complex starts prior to redistribution of plasma membrane phosphatidylserine, which serves as a signal for phagocytotic elimination of apoptotic cells. Furthermore, a caspase-resistant GFP-labelled mutant of POM121 resisted degradation even in late apoptosis and was detected in clustered nuclear pores. Thus, it can be concluded that loss of POM121-GFP is a specific sensor of the activation of caspase-3-dependent proteolysis at the nuclear pores.     

Relative quantification of membrane proteins in wild-type and prion protein (PrP)-knockout cerebellar granule neurons

Approximately 25% of eukaryotic proteins possessing homology to at least two transmembrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme α-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.     

A Leuconostoc lactis protein with homology to ribosomal protein S1 shares common epitopes and common DNA binding properties with a mammalian DNA binding nuclear factor

A mouse testis cDNA expression library (Clontech) was screened with a synthetic oligonucleotide ligand containing CT-rich motifs derived from the rat skeletal muscle actin gene promoter. These motifs bind nuclear proteins, and seem to be involved in the regulation of the gene. Analysis of isolated clones, which expressed proteins that specifically bind the oligonucleotide, indicated that they were derived from a single gene. This gene was identified as a contaminant of bacterial origin (Leuconostoc lactis). The cloned gene from L. lactis encodes a protein with significant homology to bacterial ribosomal protein S1, which we designated LrpS1-L. Band shift analysis and competition experiments indicated that both the bacterial protein and a mouse nuclear protein specifically bind to the same CT-rich motif of the skeletal muscle actin promoter. Furthermore, antibodies against the recombinant bacterial protein interfered with the formation of complex between the CT-rich element and the mouse nuclear protein. These results indicate that the bacterial LrpS1-L protein and the mammalian protein bind the same CT-rich motif and share common antigenic epitopes.     

A gene encoding a novel isoform of the PR-1 protein family from tomato is induced upon viroid infection

A Lycopersicon esculentum cDNA clone encoding an acidic-type pathogenesis-related protein (PR-lal) was isolated, sequenced and characterized. It contains an open reading frame of 175 amino acids and the mature protein, after cleavage of the 21 amino acid signals peptide, has a pl of 5.24. The protein shows highest homology (75% identity) with the basic pathogenesis-related prb-lb protein from tobacco. The PR-lal gene shows constitutive expression in roots from tomato plants. It is expressed in leaves and stems upon viroid infection, and appears to be induced by ethylene. Comparative studies of this gene and a related basic isoform of PR-1 indicate that the expression of these two members of the PR-1 gene family in tomato may be differentially regulated upon viroid infection.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X71592.     

The wheat protein kinase gene,TaPK3/, of the PKABA1 subfamily is differentially regulated in greening wheat seedlings

We have identified a new wheat PKABA1/-like protein kinase gene, TaPK3/, that is expressed in greening wheat seedlings. TaPK3 has high sequence homology (97% similarity with some sequence diversity at the 3' end) to the wheat PKABA1 protein kinase mRNA, which is upregulated by cold-temperature treatment, dehydration and abscisic acid (ABA). Use of a TaPK3 gene-specific probe has revealed that TaPK3 is differentially expressed with respect to PKABA1. TaPK3 mRNA accumulates in greening shoot tissue of wheat, but is not affected by dehydration, cold-temperature treatment or ABA. Based on sequence and expression differences, we conclude that expression of the PKABA1/-like protein kinases is not limited to stress responses.     

POM152 is an integral protein of the pore membrane domain of the yeast nuclear envelope

We have identified a concanavalin A-reactive glycoprotein of 150 kD that coenriches with isolated yeast nuclear pore complexes. Molecular cloning and sequencing of this protein revealed a single canonical transmembrane segment. Epitope tagging and localization by both immunofluorescence and immunoelectron microscopy confirmed that it is a pore membrane protein. The protein was termed POM152 (for pore membrane protein of 152 kD) on the basis of its location and cDNA-deduced molecular mass. POM152 is likely to be a type II membrane protein with its NH2-terminal region (175 residues) and its COOH-terminal region (1,142 residues) positioned on the pore side and cisternal side of the pore membrane, respectively. The proposed cisternally exposed domain contains eight repetitive motifs of approximately 24 residues. Surprisingly, POM152 deletion mutants were viable and their growth rate was indistinguishable from that of wild-type cells at temperatures between 17 and 37 degrees C. However, overproduction of POM152 inhibited cell growth. When expressed in mouse 3T3 cells, POM152 was found to be localized to the pore membrane, suggesting a conserved sorting pathway between yeast and mammals.     

Dynamic properties of nuclear pore complex proteins in gp210 deficient cells

Gp210, an integral membrane protein of the nuclear pore complex (NPC), is believed to be involved in NPC biogenesis. To test this hypothesis, we have investigated dynamic properties of the NPC and distribution of NPC proteins in NIH/3T3 cells lacking gp210. POM121 (the other integral NPC protein) and NUP107 (of the NUP107/160 complex) were correctly distributed at the nuclear pores in the absence of gp210. Furthermore, fluorescence recovery after photobleaching experiments showed that POM121 and NUP107 remained stably associated at the NPCs. We conclude that gp210 cannot be required for incorporation of POM121 or NUP107 or be required for maintaining NPC stability.     

A novel porcine gene, POT1, differentially expressed in the longissimus muscle tissues from Wujin and Large White pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus muscle tissues from Wujin and Large White pigs. One novel gene differentially expressed was identified through quantitative real time PCR and the cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 507 amino acids that shares high homology with the protection of telomeres 1 isoform 4 (POT1) of human (86%)-so that this gene can be defined as swine POT1 gene. This gene is structured in 12 exons and 11 introns as revealed by computer-assisted analysis. The tissue expression analysis indicated that the swine POT1 gene is differentially expressed in tissues including muscle, heart, liver, fat, kidney, lung, pancreas and spleen. Our experiment is the first to establish the primary foundation for further research on the swine POT1 gene.     

The Drosophila melanogaster LEM-domain protein MAN1

Here we describe the Drosophila melanogaster LEM-domain protein encoded by the annotated gene CG3167 which is the putative ortholog to vertebrate MAN1. MAN1 of Drosophila (dMAN1) and vertebrates have the following properties in common. Firstly, both molecules are integral membrane proteins of the inner nuclear membrane (INM) and share the same structural organization comprising an N-terminally located LEM motif, two transmembrane domains in the middle of the molecule, and a conserved RNA recognition motif in the C-terminal region. Secondly, dMAN1 has similar targeting domains as it has been reported for the human protein. Thirdly, immunoprecipitations with dMAN1-specific antibodies revealed that this Drosophila LEM-domain protein is contained in protein complexes together with lamins Dm0 and C. It has been previously shown that human MAN1 binds to A- and B-type lamins in vitro. During embryogenesis and early larval development LEM-domain proteins dMAN1 and otefin show the same expression pattern and are much more abundant in eggs and the first larval instar than in later larval stages and young pupae whereas the LEM-domain protein Bocksbeutel is uniformly expressed in all developmental stages. dMAN1 is detectable in the nuclear envelope of embryonic cells including the pole cells. In mitotic cells of embryos at metaphase and anaphase, LEM-domain proteins dMAN1, otefin and Bocksbeutel were predominantly localized in the region of the two spindle poles whereas the lamin B receptor and lamin Dm0 were more homogeneously distributed. Downregulation of dMAN1 by RNA interference (RNAi) in Drosophila cultured Kc167 cells has no obvious effect on nuclear architecture, viability of RNAi-treated cells and the intracellular distribution of the LEM-domain proteins Bocksbeutel and otefin. In contrast, the localization of dMAN1, Bocksbeutel and otefin at the INM is supported by lamin Dm0. We conclude that the dMAN1 protein is not a limiting component of the nuclear architecture in Drosophila cultured cells.     

Two distinct human POM121 genes: requirement for the formation of nuclear pore complexes

Pom121 is one of the integral membrane components of the nuclear pore complex (NPC) in vertebrate cells. Unlike rodent cells carrying a single POM121 gene, human cells possess multiple POM121 gene loci on chromosome 7q11.23, as a consequence of complex segmental-duplications in this region during human evolution. In HeLa cells, two "full-length" Pom121 are transcribed and translated by two distinct genetic loci. RNAi experiments showed that efficient depletion of both Pom121 proteins significantly reduces assembled NPCs on nuclear envelope. Pom121-depletion also induced clustering of NPCs, indicating its role on maintenance of NPC structure/organization.