Larry R. Gerace
Departments of Cell and Molecular Biology
Scripps Research Institute
10666 N. Torrey Pines Road
La Jolla, CA 92037
1982 Searle Scholar
The nuclear envelope is a specialized domain of the endoplasmic reticulum that forms the boundary of the nucleus in eukaryotes. It consists of inner and outer membranes, the nuclear lamina and nuclear pore complexes (NPCs). The nuclear lamina is a protein meshwork lining the inner nuclear membrane that provides a framework for the nuclear envelope and an anchoring site at the nuclear periphery for interphase chromosomes. NPCs are large supramolecular structures that span the nuclear envelope and mediate molecular transport between the nucleus and cytoplasm. We are using a combination of biochemical, structural and functional approaches to investigate the mechanisms involved in the nuclear import of protein and the role of the lamina in the nuclear organization.
Nuclear import of protein.
We found that the GTP-bound form of Ran directly interacts with a protein called RanBP2 that is localized at the cytoplasmic periphery of the NPC. This protein appears to be the site where GTP is hydrolyzed by Ran and the NLS receptor-substrate complex initially "docks" at the NPC. Our data suggest that Ran GTP hydrolysis at RanBP2 is involved in commitment of the docked substrate to downstream steps of transport. To understand this in detail, we are investigating the protein that activates Ran GTPase and other factors that interact with RanBP2.
After docking at the periphery of the NPC, the NLS receptor-substrate complex is moved 50-100 nm to accumulate near the central gated channel. This movement may involve Tpr, a large coiled-coil protein we recently identified that may provide an anchoring site for RanBP2. The substrate collection site near the central channel is likely to be the p62 complex, a protein of about 234 kD protein consisting of p62, p58, p54 and p45. Using electron microscopy, we found that the isolated p62 complex has an intriguing donut-shaped structure, compatible with the notion that it interacts with the central channel. Results of cDNA cloning showed that the p58 and p45 subunits contain multiple "FG" dipeptide motifs, similar to the previously characterized p62 subunit. We have determined that multiple cytosolic transport factors including NTF2 interact with the p62 complex. NTF2 directly binds to p62 and also specifically associates with the GDP-bound form of Ran. This finding suggests that NTF2 functions as an adaptor to target a "committed" NLS receptor-substrate complex to the p62 complex.
Nuclear lamina and chromosome organization.
We also have found that nuclear lamins as well as LAP2 specifically interact with chromatin with moderately high affinity. We determined that the chromatin components interacting with lamins are core histone, and have mapped the histone-binding region of A-type lamins to a short segment of the carboxyl terminal "tail" domain. These and other data indicate that the nuclear lamina engages in a complex set of cooperative, developmentally regulated interactions with chromatin that involves both lamins and integral membrane proteins, which could be important for stabilizing or modifying patterns of gene expression in cells.
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