News and Publications

Nucleocytoplasmic Transport and Role of the Nuclear Lamina in Higher Level Nuclear Organization

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.

Nucleocytoplasmic Transport Mechanisms

Transport of most proteins through the NPC is an energy-dependent process mediated by nucleocytoplasmic shuttling receptors of the importin ß superfamily. These receptors bind to specific transport signals on cargo molecules and are translocated through the NPC by interaction of the receptors with NPC proteins (nucleoporins) that contain 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 events that specify transport of cargo-receptor complexes through the NPC. Our results support a model in which movement of cargo-receptor complexes occurs by facilitated diffusion though a dense meshwork formed by phenylalanine-glycine repeat nucleoporins. This process appears to be promoted by Ran-GTP-mediated dissociation of receptors from nucleoporins and by an increase in the binding affinity of receptor-cargo complexes for nucleoporins at progressively more distal sites in the transport pathway.

We defined several critical tryptophan residues involved in the binding of the adaptor importin α to the receptor importin ß; the binding may trigger a mutually induced conformational change in the adaptor and the receptor upon their association. Using x-ray crystallography, we solved the structure of a fragment of importin ß complexed with parathyroid hormone-related protein, which represents a novel class of cargo. Unlike importin α, 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 supports import of parathyroid hormone-related protein in vitro, it may be a prototypical nuclear transport receptor. Consistent with this prediction, we identified a nucleoporin binding site in the carboxyl-terminal half of importin ß that contributes strongly to the import function of importin ß.

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 viral docking and uncoating at the NPC and subsequent translocation of viral DNA into the nucleus. Whereas the major capsid protein, hexon, appears to be involved in viral 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.

In an analysis of the nuclear import of hexon, we found that protein VI, another viral structural protein, acts as a nuclear-cytoplasmic shuttling adapter for hexon import. Interestingly, the carboxyl-terminal sequence of protein VI that mediates shuttling is cleaved by proteolysis at the time of viral maturation, an example of regulation of protein function by cleavable nuclear transport signals.

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. The 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 lamina-associated polypeptides 1 and 2. Recent findings support the notion that the lamina is a key component for determining nuclear shape and increase in surface area during the cell cycle.

In related studies, we are analyzing the role of the lamina in the anchoring of chromosomes to the nuclear periphery in the interphase nucleus and the importance of this interaction for regulation of chromatin activity. Our results indicate that the attachment of chromatin to the nuclear envelope involves both lamins and integral membrane protein, which interact with qualitatively distinct chromosomal components. In a recent proteomic analysis, we identified 57 novel polypeptides that are putative components of the inner nuclear membrane, indicating that the inner membrane has an unexpectedly complex protein composition. The importance of lamins and inner membrane proteins in chromatin function is underscored by the finding that an increasing number of human diseases have been linked to defects in components of the lamina.

Publications

Bednenko, J., Cingolani, G., Gerace, L. Nucleocytoplasmic transport: navigating the channel. Traffic 4:127, 2003.

Cingolani, G., Bednenko, J., Gillespie, M.T., Gerace, L. Molecular basis for the recognition of a nonclassical nuclear localization signal by importin ß. Mol. Cell 10:1345, 2002.

Cingolani, G., Gerace, L. Molecular basis for nucleocytoplasmic transport. In: Handbook of Cell Signaling. Bradshaw, R.A., Dennis, E.A. (Eds.). Academic Press, San Diego, in press.

Kehlenbach, R.H., Gerace, L. Analysis of nuclear protein import and export in vitro using fluorescent cargoes. Methods Mol. Biol. 189:231, 2002.

Koerner, C., Guan, T., Gerace, L, Cingolani, G. Synergy of silent and hot spot mutations in importin ß reveals a dynamic mechanism for recognition of a nuclear localization signal. J. Biol. Chem. 278:16216, 2003.