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Nuclear-Cytoplasmic Transport and Role of the Nuclear Lamina in Higher Level Nuclear Organization

J. Bednenko, I. Ben-Efraim, G. Cingolani, P. Frosst, L. Gerace, T. Guan, A. Kehlenbach, R. Kehlenbach, S. Lyman, E. Schirmer, I. Schmitt, H. Wodrich, T. Ohba

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 provide channels for 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 most proteins through the NPC is an energy-dependent process mediated by nucleocytoplasmic shuttling receptors of the importin ß/karyopherin ß family. These receptors bind to specific transport signals on cargo molecules and are translocated through the NPC by a multistep process that involves interaction of the receptors with members of a group of NPC proteins (nucleoporins) containing multiple repeats of a phenylalanine-glycine di-amino acid motif. A key regulator of many nuclear transport pathways is the small GTPase Ran, which promotes binding of cargo to export receptors and dissociation of cargo from import receptors in the nucleus.

We are using in vitro assays and digitonin-permeabilized cells to analyze the molecular components and events that specify translocation of cargo-receptor complexes through the NPC. We found that an increase in the binding affinity of receptor-cargo complexes for nucleoporins at progressively more distal sites in the transport pathway is important for promoting directional movement of import and export receptors through the pore. We are using x-ray crystallography and site-directed mutagenesis to obtain a detailed structural understanding of the interaction of the import receptor importin ß with specific nucleoporins containing phenylalanine-glycine repeats and cargoes.

We recently solved the structure of a fragment of importin ß complexed with parathyroid hormone--related protein, which represents a novel class of transport cargo. Unlike cargo-binding adaptors such as importin a, which interacts with the carboxyl-terminal part of importin ß, parathyroid hormone--related protein binds to the amino-terminal half of importin ß. Because this fragment also contains binding sites for both Ran and nucleoporins and can support import of parathyroid hormone--related protein in vitro, it may resemble a prototypical nuclear transport receptor.

To define principles used for the transport of viral genomes through the NPC, we are analyzing the nuclear import of adenovirus DNA. Our findings suggest that this process is driven by adenovirus DNA--associated proteins, which interact with the nuclear import machinery to potentiate virus uncoating and translocation through the NPC. We are also investigating the export of mRNA-protein complexes from the nucleus. For these studies, we developed an in vitro model to examine the nuclear export of TAP, a protein that binds to and promotes export of certain viral and cellular mRNAs. Surprisingly, we found that the nuclear transport sequence of TAP mediates export via direct interactions with nucleoporins, without the intervention of karyopherin ß--like receptors or Ran. This finding may reflect a theme for nuclear export of many mRNAs.

NUCLEAR LAMINA AND HIGHER LEVEL NUCLEAR ORGANIZATION

The nuclear lamina of higher eukaryotes consists of 2--4 related intermediate filament proteins called lamins and a number of more minor lamina-associated polypeptides (LAPs). Attachment of the lamina to the inner nuclear membrane during interphase and reassembly of the nuclear envelope at the end of mitosis appear to involve interactions between lamins and integral membrane proteins of the inner nuclear membrane, including LAP1 and LAP2. The results of recent studies support the notion that the lamina is key to higher level nuclear organization. We found that when expressed in cultured cells, certain lamin mutants lead to gross disruption of nuclear shape and production of micronuclei. Moreover, injection of the lamin-binding region of LAP2 into mitotic or G₁ phase cells selectively blocks nuclear growth but does not otherwise perturb functions of the nuclear envelope. These findings indicate that the growth of the lamina regulates changes in nuclear volume during the cell cycle. Because entry into S phase is also inhibited in this condition, these data suggest the existence of a checkpoint control linking nuclear volume to activation of DNA replication.

In related studies, we are analyzing the role of the lamina in the anchoring of chromosomes within the 3-dimensional space of the interphase nucleus and the importance of this interaction for chromosome functions. Our results indicate that the attachment of chromatin to the nuclear envelope involves both lamins and LAP2, which interact with qualitatively distinct chromosomal components that could be independent targets of regulation. Using antibodies to inhibit the lamin-chromatin interaction in vivo, we obtained direct evidence that the association of chromatin with the lamina is important for proper DNA replication.

PUBLICATIONS
Ben-Efraim, I., Gerace, L. Gradient of increasing affinity of importin ß for nucleoporins along the pathway of nuclear import. J. Cell Biol. 152:411, 2001.

Cingolani, G., Lashuel, H.A., Gerace, L., Muller, C.W. Nuclear import factors importin a and importin ß undergo mutually induced conformational changes upon association. FEBS Lett. 484:291, 2001.

Guan, T., Kehlenbach, R.H., Schirmer, E.C., Kehlenbach, A., Fan, F., Clurman, B.E., Arnheim, N., Gerace, L. Nup50, a nucleoplasmically oriented nucleoporin with a role in nuclear protein export. Mol. Cell. Biol. 20:5619, 2000.

Kehlenbach, R.H., Assheuer, R., Kehlenbach, A., Becker, J., Gerace, L. Stimulation of nuclear export and inhibition of nuclear import by a Ran mutant deficient in binding to Ran-binding protein 1. J. Biol. Chem. 276:14524, 2001.

Schirmer, E.C., Guan, T., Gerace, L. Involvement of the lamin rod domain in heterotypic lamin interactions important for nuclear organization. J. Cell Biol. 153:479, 2001

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