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

L. Gerace, J. Bednenko, I. Ben-Efraim, G. Cingolani, P. Frosst, T. Guan, S. Lyman, T. Oba, E. Schirmer, H. Wodrich, C. Koerner, T. Palmer

The nuclear envelope is a specialized domain of the endoplasmic reticulum that forms the boundary of the nucleus in eukaryotic cells. The envelope 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/karyopherin b family. These receptors bind to specific transport signals on cargo molecules and are translocated through the NPC by a multistep process that involves the interaction of the receptors with members of a group of NPC proteins (nucleoporins) containing multiple repeats of a phenylalanine-glycine 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 with digitonin-permeabilized cells to analyze the molecular components and events that specify import of cargo-receptor complexes into the nucleus. Our results support a model in which movement of cargo-receptor complexes through the NPC occurs by facilitated diffusion via interactions with a group of phenylalanine-glycine repeat nucleoporins. This process appears to be facilitated by Ran-GTP-mediated dissociation of receptors from nucleoporins and an increase in the binding affinity of receptor-cargo complexes for nucleoporins at progressively more distal sites in the transport pathway.

We are using x-ray crystallography and site-directed mutagenesis to obtain a detailed structural understanding of the interaction of the import receptor importin b with specific phenylalanine-glycine repeat nucleoporins and cargoes. We defined critical tryptophan residues involved in the binding of the adaptor importin a to importin b the binding may trigger a mutually induced conformational change in the receptor and adaptor upon their association. We also solved the structure of a fragment of importin b complexed with parathyroid hormone-related protein, which represents a novel class of cargo. Unlike importin a

To define principles used for the transport of viral genomes through the NPC, we are analyzing the nuclear import of adenovirus DNA. Our results suggest that this process is driven by adenoviral proteins, which interact with the nuclear import machinery to potentiate docking and uncoating of the virus at the NPC and subsequent translocation of viral DNA into the nucleus. Whereas the major capsid protein, hexon, appears to be involved in virus docking at the NPC, our data indicate that protein VII, the major DNA-packaging protein of adenovirus, contains multiple nuclear import signals that may drive the nuclear transport of the associated DNA.

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.

Recent findings support the notion that the lamina is key to higher level nuclear organization. Expression of certain lamin mutants in cultured cells led 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 blocked nuclear growth but did 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.

PUBLICATIONS

Cingolani, G., Bednenko, J., Gillespie, M.T., Gerace, L. Molecular basis for the recognition of a nonclassical nuclear localization signal by importin b. Mol. Cell, in press.

Frosst, P., Guan, T., Subauste, C., Hahn, K., Gerace, L. Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export. J. Cell Biol. 156:617, 2002.

Lyman, S.K., Gerace, L. Nuclear pore complexes: dynamics in unexpected places. J. Cell Biol. 154:17, 2001.

Lyman, S.K., Guan, T., Bednenko, J., Wodrich, H., Gerace, L. Influence of cargo size on Ran and energy requirements for nuclear protein import. J. Cell Biol. 159:55, 2002.

Schirmer, E.C., Gerace, L. Organellar proteomics: the prizes and pitfalls of opening the nuclear envelope. Genome Biol. 3:REVIEWS1008.1, 2002.

Schmitt, I., Gerace, L. In vitro analysis of nuclear transport mediated by the C-terminal shuttle domain of Tap. J. Biol. Chem. 276:42355, 2001.