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Nuclear-Cytoplasmic Transport and Higher Level Nuclear Organization

I. Ben-Efraim, C. Delphin, A. Dickmanns, C. Fritze, P. Frosst, L. Gerace, T. Guan, R. Kehlenbach, S. Lyman, R. Mahajan, F. Melchior, A. Saphire, I. Schmitt, L. Yang

The nuclear envelope is a specialized domain of the endoplasmic reticulum that forms the boundary of the eukaryotic cell nucleus. It consists of inner and outer nuclear membranes, the nuclear lamina, and nuclear pore complexes (NPCs). The nuclear lamina, a protein meshwork lining the inner nuclear membrane, is thought to provide a framework for the nuclear envelope and an anchoring site at the nuclear periphery for interphase chromosomes. NPCs are large supramolecular assemblies that span the nuclear envelope and mediate molecular transport between the nucleus and the cytoplasm. We are using biochemical, structural, and functional approaches to investigate the functions of NPCs and the lamina.

NUCLEAR-CYTOPLASMIC TRANSPORT MECHANISMS

Transport of protein and RNA through the NPC involves energy- and signal-dependent mechanisms. We are using a cytosol-dependent in vitro assay with digitonin-permeabilized cells to investigate the nuclear import of proteins. Nuclear import specified by nuclear localization sequences (NLSs) rich in basic amino acids requires multiple cytosolic factors, including importins and ß, NTF2, and the small GTPase Ran. Importins and ß form a complex that functions as a shuttling carrier for the nuclear import of NLS-containing molecules. During the course of its movement to the nuclear interior, the importin-containing transport complex sequentially interacts with several NPC regions, including peripheral and internal NPC-binding sites and a central gated channel.

A group of NPC proteins containing multiple repeats of a phenylalanine-glycine dipeptide motif appears to provide binding sites for the importin /ß transport complex during transit of the complex through the NPC. We are analyzing several of these proteins in detail, including RanBP2 and a 4-subunit protein called the p62 complex. Our data indicate that RanBP2 provides an initial binding site for the substrate-importin complex at the NPC periphery. We found that the GTPase-activating protein for Ran, RanGAP1, is tightly complexed with RanBP2 and that hydrolysis of Ran-GTP at this site is required for nuclear import. This GTP hydrolysis step may commit the import complex to downstream transport steps.

We determined that the targeting of RanGAP1 to RanBP2 is mediated by a novel ubiquitin-related polypeptide, SUMO-1, that is covalently coupled to RanGAP1. The attachment of SUMO-1 to RanGAP1 is reversible, and this reversible attachment potentially could provide a mechanism to regulate nuclear transport. Further movement of the transport complex through the NPC is promoted by the cytosolic factor NTF2, which specifically interacts with subunits of the p62 complex and with GDP-Ran. Our data suggest that the p62 complex is a collection site near the center of the NPC for the substrate-NLS receptor complex that is involved in transferring the latter complex to the central gated channel of the NPC.

We also developed permeabilized cell assays to study signal-mediated export of nuclear proteins. We showed that in vitro export of the transcription factor NF-AT requires several cytosolic factors, including Ran and an export receptor related to importin ß, that shuttle between the nucleus and the cytoplasm. This permeabilized cell system provides us with an easy biochemical assay to directly characterize components of the nuclear export machinery. Using purified adenovirions, we have also reconstituted in vitro the nuclear import of adenovirus DNA. This system should be useful for understanding the general problem of nucleic acid transport through the NPC.

NUCLEAR LAMINA AND HIGHER LEVEL NUCLEAR ORGANIZATION

The nuclear lamina of higher eukaryotes consists mainly of 2--4 related intermediate filament proteins called lamins. To understand the role of the lamina in nuclear organization, we are characterizing its interactions with the inner nuclear membrane and chromosomes. Our data indicate that the attachment of chromatin to the nuclear envelope involves multiple lamina proteins, including lamins and LAP2. Collectively, by regulating the positioning of chromosomes in the nucleus and thereby affecting their transcriptional activity, these interactions could be important for stabilizing or modifying patterns of gene expression in cells.

Both attachment of the lamina to the inner nuclear membrane during interphase and reassembly of the nuclear envelope at the end of mitosis involve interactions between lamins and integral membrane proteins of the nuclear envelope, particularly LAP1 and LAP2. We found that the LAPs become randomly dispersed throughout the endoplasmic reticulum after disassembly of the nuclear envelope in mitosis, indicating that the nuclear envelope loses its identity as a discrete subcompartment of the endoplasmic reticulum during this period. This finding suggests that LAPs reassemble in the nuclear envelope at the end of mitosis by diffusing through fused membranes of the endoplasmic reticulum and binding to lamins and other components at the chromosome surfaces. By injecting the lamin-binding region of LAP2 into mitotic or G₁-phase cells, we can selectively inhibit nuclear growth and entry into S phase; assembly of the nuclear envelope and nuclear transport are unaffected. These data provide direct evidence that the nuclear lamina is involved in the increase in nuclear volume that occurs during the cell cycle and, correspondingly, in activation of DNA replication.

PUBLICATIONS

Furukawa, K., Fritze, C.E., Gerace, L. The major nuclear envelope targeting domain of LAP2 coincides with its lamin binding region but is distinct from its chromatin interaction domain. J. Biol. Chem. 273:4213, 1998.

Hu, T., Gerace, L. cDNA cloning and analysis of the expression of nucleoporin p45. Gene, in press.

Kehlenbach, R.H., Dickmanns, A., Gerace, L. Nucleocytoplasmic shuttling factors including Ran and CRM1 mediate nuclear export of NFAT in vitro. J. Cell Biol. 141:863, 1998.

Mahajan, R., Gerace, L., Melchior, F. The C-terminal domain of RanGAP1 contains the acceptor site for SUMO1 modification and is sufficient for targeting to the nuclear envelope. J. Cell Biol. 140:259, 1998.

Melchior, F., Gerace, L. Two-way trafficking with Ran. Trends Cell Biol. 8:175, 1998.

Saphire, A.C.S., Bark, S.J., Gerace, L. All four homochiral enantiomers of a nuclear localization sequence derived brom c-Myc serve as functional import signals. J. Biol. Chem., in press.

Westendorf, J.M., Konstantinov, K.M., Wormsley, S., Shu, M.-D., Matsumoto-Taniura, N., Pirollet, F., Klier, F.G., Gerace, L., Baserga, S.J. M-phase phosphoprotein 10 (MPP10), is a U3snoRNP-specific nucleolar protein. Mol. Biol. Cell 9:437, 1998.