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We have developed an in vitro system involving digitonin-permeabilized vertebrate cells to study biochemical events in the transport of macromolecules across the nuclear envelope. While treatment of cultured cells with digitonin permeabilizes the plasma membranes to macromolecules, the nuclear envelopes remain structurally intact and nuclei retain the(More)
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(More)
We have investigated a possible involvement of GTPases in nuclear protein import using an in vitro transport system involving digitonin-permeabilized cells supplemented with exogenous cytosol. Transport in this system was measured with a novel ELISA-based assay that allows rapid quantitative analysis. GTP gamma S and other nonhydrolyzable analogues of GTP(More)
Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an(More)
Lamina-associated polypeptides (LAPs) 1A, 1B, 1C, and 2 are integral membrane proteins of the nuclear envelope associated with the nuclear lamina. Using in vitro assays, we show that LAPs 1A and 1B specifically bind to both lamins A and C and lamin B1, while LAP 2 associates only with lamin B1. LAP 2 also binds to mitotic chromosomes. The LAPs are(More)
We described a cell free system involving total homogenates of metaphase CHO cells, which yields telophase-like assembly of nuclear envelopes around mitotic chromosomes. During formation of the nuclear envelope in vitro, the three major lamina polypeptides (lamins A, B, and C) assemble around chromosomes and become dephosphorylated, similar to their(More)
The nuclear envelope lamina is a supramolecular protein assembly associated with the nucleoplasmic surface of the inner nuclear membrane, which contains three predominant polypeptide components in mammalian cells (lamins A, B and C). We previously demonstrated by immunofluorescence microscopy that the lamina is reversibly disassembled during cell division,(More)
The nuclear lamina, a protein meshwork lining the nucleoplasmic surface of the inner nuclear membrane, is thought to provide a framework for organizing nuclear envelope structure and an anchoring site at the nuclear periphery for interphase chromatin. In several higher eukaryotic cells, the lamina appears to be a polymer comprised mainly of one to three(More)
To comprehensively identify integral membrane proteins of the nuclear envelope (NE), we prepared separately NEs and organelles known to cofractionate with them from liver. Proteins detected by multidimensional protein identification technology in the cofractionating organelles were subtracted from the NE data set. In addition to all 13 known NE integral(More)
We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that(More)