The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) - the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) - ...a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.
One of the defining features of eukaryotic cells is the segregation of RNA biogenesis and DNA replication in the nucleus, separate from the cytoplasmic machinery for protein synthesis. Integration of ...the activities of the nucleus and cytoplasm requires the continuous transport of proteins, RNAs, and small molecules between these two compartments. Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs), large supramolecular structures that span the nuclear envelope. NPCs contain aqueous channels with a diameter of similar to 10 nm, which allow ions, metabolites, and small proteins to diffuse passively between the nucleus and cytoplasm. Most proteins and RNAs are too large to cross the NPC by passive diffusion at physiologically relevant rates. Instead, they are transported through a gated transport channel in the NPC by active mechanisms, which are saturable and energy dependent and involve specific signals on the transported molecules. While substantial insight into the molecular basis for nuclear protein import has been obtained recently, nuclear export remains poorly understood. A watershed for the analysis of nuclear protein import was the discovery that short amino acid stretches termed nuclear localization signals (NLSs) specify the nuclear import of many proteins. While NLSs lack a strict consensus, they are usually highly enriched in basic amino acids. NLSs containing both single and bipartite stretches of basic residues have been described. These NLSs can function when transplanted to nonnuclear proteins, either by inserting NLS-specifying codons into cDNAs or by coupling synthetic peptides containing NLSs to folded proteins. Characterization of nuclear export signals (NESs) has lagged considerably behind analysis of NLSs. This partly is due to the experimental difficulty of studying nuclear export, but also relates to the complex nature of many of the molecular species that are exported from the nucleus. The RNA substrates for nuclear export are probably all transported as RNA-protein (RNP) complexes. Many or all of these RNPs contain multiple polypeptides, so it is difficult to know the precise composition of the substrate that is actually transported. Moreover, it is not known whether the molecular signals that interact with the nuclear transport machinery reside in the RNAs themselves or in the proteins that are bound to the RNAs. A new perspective on the problem of nuclear export emerged recently with the characterization of specific proteins that rapidly shuttle between the nucleus and cytoplasm, which have the potential to bear NESs as well as import signals. These shuttling species include RNA-binding proteins, such as the hnRNP A1 protein and the HIV-1 Rev protein, as well as proteins that have no obvious interaction with RNAs, including certain transcriptional activators and hsc70. A detailed analysis of the nuclear trafficking pathways of two shuttling proteins, the cAMP-dependent protein kinase (PKA) and Rev, has now led to the definition of short protein sequences responsible for rapid nuclear export.
Mutations in certain nuclear envelope (NE) proteins have been shown to cause a diversity of human diseases, including muscular dystrophy, lipdystrophy, and the premature aging condition, progeria. ...New research into torsinA, a product of the DYT1 gene, is mutated in early-onset torsion dystonia, functions in NE and may possibly cause the disease.
Nucleocytoplasmic transport is mediated by shuttling receptors that recognize specific signals on protein or RNA cargoes and translocate the cargoes through the nuclear pore complex. Transport ...receptors appear to move through the nuclear pore complex by facilitated diffusion, involving repeated cycles of binding to and dissociation from nucleoporins with phenylalanine‐glycine motifs. We discuss recent experimental approaches and results that have begun to provide molecular insight into the mechanisms by which transport complexes traverse the nuclear pore complex, and point out the significant gaps in understanding that remain.
We have found that the mammalian Ran GTPase–activating protein RanGAP1 is highly concentrated at the cytoplasmic periphery of the nuclear pore complex (NPC), where it associates with the 358-kDa ...Ran–GTP-binding protein RanBP2. This interaction requires the ATP-dependent posttranslational conjugation of RanGAP1 with SUMO-1 (for small ubiquitin-related modifier), a novel protein of 101 amino acids that contains low but significant homology to ubiquitin. SUMO-1 appears to represent the prototype for a novel family of ubiquitin-related protein modifiers. Inhibition of nuclear protein import resulting from antibodies directed at NPC-associated RanGAP1 cannot be overcome by soluble cytosolic RanGAP1, indicating that GTP hydrolysis by Ran at RanBP2 is required for nuclear protein import.
The nuclear lamina, which provides a structural scaffolding for the nuclear envelope, consists largely of a polymer of the
intermediate filament lamin proteins. Although different cell types contain ...distinctive relative amounts of the major lamin
subtypes (A, C, B1, and B2), the functions of this variation are not understood. We have investigated the possibility that
subtype variation affects lamina stability. We find that homotypic and heterotypic binding interactions of lamin B2 are substantially
less resistant to chemical dissociation in vitro than those between the other lamin subtypes, whereas lamin A interactions are the most stable. Surprisingly, removal of the
central four-fifths of the rod domain did not substantially weaken the interactions of lamins A and B2, suggesting that other
regions also strongly contribute to their binding interactions. In contrast, this rod deletion strongly destabilizes the binding
interactions of lamins B1 and C. Consistent with the binding studies, lamins are more readily solubilized by chemical extraction
from cells enriched for lamin B2 than from cells enriched for lamin A. This suggests that the distinctive ensemble of heterotypic
lamin interactions in a particular cell type affects the stability of the lamin polymer, and, correspondingly, could be relevant
to tissue-specific properties of the lamina including its involvement in disease.
The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, ...mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101- amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1-SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.
Tpr is a coiled-coil protein found near the nucleoplasmic side of the pore complex. Since neither the precise localization of Tpr nor its functions are well defined, we generated antibodies to three ...regions of Tpr to clarify these issues. Using light and EM immunolocalization, we determined that mammalian Tpr is concentrated within the nuclear basket of the pore complex in a distribution similar to Nup153 and Nup98. Antibody localization together with imaging of GFP-Tpr in living cells revealed that Tpr is in discrete foci inside the nucleus similar to several other nucleoporins but is not present in intranuclear filamentous networks (Zimowska et al., 1997) or in long filaments extending from the pore complex (Cordes et al., 1997) as proposed. Injection of anti-Tpr antibodies into mitotic cells resulted in depletion of Tpr from the nuclear envelope without loss of other pore complex basket proteins. Whereas nuclear import mediated by a basic amino acid signal was unaffected, nuclear export mediated by a leucine-rich signal was retarded significantly. Nuclear injection of anti-Tpr antibodies in interphase cells similarly yielded inhibition of protein export but not import. These results indicate that Tpr is a nucleoporin of the nuclear basket with a role in nuclear protein export.
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