The Tap protein of higher eukaryotes is implicated in the nuclear export of type D retroviral mRNA and some cellular mRNAs. Here we have developed an in vitroassay to study nuclear export mediated by ...the C-terminal shuttle domain of Tap involving the rapamycin-induced attachment of this transport domain to a nuclear green fluorescent protein-containing reporter. We found that export by the Tap transport domain does not involve cytosolic transport factors including the GTPase Ran. The transport domain directly binds to several nucleoporins positioned in different regions of the nuclear pore complex. These results argue that a direct interaction of the Tap transport domain with nucleoporins is responsible for its nucleocytoplasmic translocation. We found that the karyopherin β-related export receptor CRM1 competes with the Tap transport domain for binding to Nup214 but not for binding to Nup62 or Nup153, suggesting that the Tap and CRM1 nuclear export pathways converge at the cytoplasmic periphery of the nuclear pore complex. Because the rates of in vitro nuclear import and export by the Tap transport domain are very similar, the directionality of mRNA export mediated by Tap probably is determined by mechanisms other than simple binding of the Tap transport domain to nucleoporins.
LAP2 is an integral protein of the inner nuclear membrane which binds lamins and chromosomes and is suggested to have an important
role in nuclear envelope organization. In a previous study we ...identified an internal 76-amino acid region of LAP2 which is
required for stable targeting of the protein to the nuclear envelope. Here, we have mapped the lamin binding region of LAP2
and demonstrate that it coincides with this nuclear envelope targeting domain. In contrast, we found that the portion of LAP2
involved in binding to chromosomes resides in a separate region of the protein near its NH 2 terminus. The minimal lamin binding region of LAP2 is capable of conferring stable nuclear envelope localization when attached
to the transmembrane and partial lumenal domains of a protein that shows no nuclear envelope targeting activity. This directly
supports the notion that a major mechanism for localization of integral membrane proteins at the inner nuclear membrane involves
binding to lamins, which would constrain diffusion through the continuous nuclear envelope/endoplasmic reticulum membrane
system.
Cellular cholesterol levels are controlled by endoplasmic reticulum (ER) sterol sensing proteins, which include Scap and Insig-1. With cholesterol sufficiency, Insig inhibits the activation of sterol ...regulatory element binding proteins (SREBPs), key transcription factors for cholesterol and fatty acid biosynthetic genes, by associating with Scap-SREBP complexes to promote their ER retention. Here we show that the multimeric ER proteins erlins-1 and -2 are additional SREBP regulators. Depletion of erlins from cells grown with sterol sufficiency led to canonical activation of SREBPs and their target genes. Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins. Erlins bound cholesterol with specificity and strong cooperativity and responded to ER cholesterol changes with altered diffusional mobility, suggesting that erlins themselves may be regulated by cholesterol. Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery. We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.
The nuclear pore complex is the gateway for protein and RNA transport between the cytoplasm and nucleus. Recent work has characterized signals and components involved in nuclear import of ...macromolecules and has described mechanisms for transport regulation. Advances in understanding the structure of the pore complex are starting to provide a framework for interpreting the biochemistry of nuclear import. Information on the export of RNA from the nucleus is only beginning to emerge.
In this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the ...nucleus. We used RNA interference (RNAi) to deplete cells of either Nup214 or Nup358, the two major Phe-Gly (FG) repeat nucleoporins localized on the cytoplasmic side of the NPC, and evaluated the impact on hexon binding and AdV infection. The accumulation of purified hexon trimers or partially disassembled AdV at the nuclear envelope (NE) was observed in digitonin-permeabilized cells in the absence of cytosolic factors. Both in vitro hexon binding and in vivo nuclear import of the AdV genome were strongly reduced in Nup214-depleted cells but still occurred in Nup358-depleted cells, suggesting that Nup214 is a major binding site of AdV during infection. The expression of an NPC-targeted N-terminal domain of Nup214 in Nup214-depleted cells restored the binding of hexon at the NE and the nuclear import of protein VII (pVII), indicating that this region is sufficient to allow AdV binding. We further narrowed the binding site to a 137-amino-acid segment in the N-terminal domain of Nup214. Together, our results have identified a specific region within the N terminus of Nup214 that acts as a direct NPC binding site for AdV.
AdVs, which have the largest genome of nonenveloped DNA viruses, are being extensively explored for use in gene therapy, especially in alternative treatments for cancers that are refractory to traditional therapies. In this study, we characterized the molecular basis for binding of AdV to the cytoplasmic face of the NPC, a key step for delivery of the viral genome into the nucleus. Our data indicate that a 137-amino-acid region of the nucleoporin Nup214 is a binding site for the major AdV capsid protein, hexon, and that this interaction is required for viral DNA import. These findings provide additional insight on how AdV exploits the nuclear transport machinery for infection. The results could promote the development of new strategies for gene transfer and enhance understanding of the nuclear import of other viral DNA genomes, such as those of papillomavirus or hepatitis B virus that induce specific cancers.
Lamina-associated polypeptides 1A-1C (LAPs1A-1C) are related integral membrane proteins of the inner nuclear membrane that bind to both A- and B-type lamins and have a putative role in the membrane ...attachment and assembly of the nuclear lamina. In this study, we have cloned a cDNA encoding LAP1C. The DNA sequence predicts a 506-amino acid protein of largely hydrophilic character with a single membrane-spanning region between residues 311-333. Mapping of the epitope recognized by the anti-LAP1 monoclonal antibody RL13 indicates that the hydrophilic domain containing residues 1-310 is exposed to the nucleoplasm and thus that LAP1C is a type II integral membrane protein. A second class of LAP1 cDNAs was isolated that contains two protein-coding nucleotide insertions in the LAP1C sequence. These probably encode parts of LAPs1A and/or −1B, suggesting that LAP1 isotypes arise from alternative splicing. Immunoblot analysis of mouse P19 teratocarcinoma cells and the P19MES-differentiated derivative of the latter suggest that LAP1 isotypes are differentially expressed during development, similar to members of the nuclear lamin family. Since the different LAP1 isotypes appear to bind lamins with different affinities, these changes in expression could be important for developmentally regulated alterations in nuclear structure.
Abstract
Aims
Nuclear envelope integrity is essential for the compartmentalization of the nucleus and cytoplasm. Importantly, mutations in genes encoding nuclear envelope (NE) and associated proteins ...are the second highest cause of familial dilated cardiomyopathy. One such NE protein that causes cardiomyopathy in humans and affects mouse heart development is Lem2. However, its role in the heart remains poorly understood.
Methods and results
We generated mice in which Lem2 was specifically ablated either in embryonic cardiomyocytes (Lem2 cKO) or in adult cardiomyocytes (Lem2 iCKO) and carried out detailed physiological, tissue, and cellular analyses. High-resolution episcopic microscopy was used for three-dimensional reconstructions and detailed morphological analyses. RNA-sequencing and immunofluorescence identified altered pathways and cellular phenotypes, and cardiomyocytes were isolated to interrogate nuclear integrity in more detail. In addition, echocardiography provided a physiological assessment of Lem2 iCKO adult mice. We found that Lem2 was essential for cardiac development, and hearts from Lem2 cKO mice were morphologically and transcriptionally underdeveloped. Lem2 cKO hearts displayed high levels of DNA damage, nuclear rupture, and apoptosis. Crucially, we found that these defects were driven by muscle contraction as they were ameliorated by inhibiting myosin contraction and L-type calcium channels. Conversely, reducing Lem2 levels to ∼45% in adult cardiomyocytes did not lead to overt cardiac dysfunction up to 18 months of age.
Conclusions
Our data suggest that Lem2 is critical for integrity at the nascent NE in foetal hearts, and protects the nucleus from the mechanical forces of muscle contraction. In contrast, the adult heart is not detectably affected by partial Lem2 depletion, perhaps owing to a more established NE and increased adaptation to mechanical stress. Taken together, these data provide insights into mechanisms underlying cardiomyopathy in patients with mutations in Lem2 and cardio-laminopathies in general.
Graphical Abstract
Graphical Abstract
Lipin-1 is a protein that has dual functions as a phosphatidic acid phosphohydrolase (PAP) and a nuclear transcriptional coactivator. It remains unknown how the nuclear localization and coactivator ...functions of lipin-1 are regulated. Here, we show that lipin-1 (including both the alpha and beta isoforms) is modified by sumoylation at two consensus sumoylation sites. We are unable to detect sumoylation of the related proteins lipin-2 and lipin-3. Lipin-1 is sumoylated at relatively high levels in brain, where lipin-1alpha is the predominant form. In cultured embryonic cortical neurons and SH-SY5Y neuronal cells, ectopically expressed lipin-1alpha is localized in both the nucleus and the cytoplasm, and the nuclear localization is abrogated by mutating the consensus sumyolation motifs. The sumoylation site mutant of lipin-1alpha loses the capacity to coactivate the transcriptional (co-) activators PGC-1alpha and MEF2, consistent with its nuclear exclusion. Thus, these results show that sumoylation facilitates the nuclear localization and transcriptional coactivator behavior of lipin-1alpha that we observe in cultured neuronal cells, and suggest that lipin-1alpha may act as a sumoylation-regulated transcriptional coactivator in brain.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The marriage of proteomics with cell biology has produced extensive inventories of the proteins that inhabit several subcellular organelles. Recent proteomic analysis has identified many new putative ...transmembrane proteins in the nuclear envelope, and transcriptome profiling suggests that the nuclear-membrane proteome exhibits some significant variations among different tissues. Cell-type-specific differences in the composition of protein sub-complexes of the nuclear envelope, particularly those containing the disease-associated protein lamin A, could yield distinctive functions and, thus, explain the tissue specificity of a diverse group of nuclear-envelope-linked disorders in humans. Considered together, these recent results suggest an unexpected functional complexity at the nuclear envelope.
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