Larry R. Gerace
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.
Nuclear import of most proteins is an active process mediated by specific amino acid sequences called nuclear localization signals (NLSs). We have developed an in vitro assay based on digitonin permeabilized cells to study the mechanisms of NLS-mediated nuclear protein import. Transport in this system depends on multiple cytosolic factors that act in concert with specific proteins in the NPC. We have identified three cytosolic factors involved in nuclear import: the NLS receptors, the small GTPases Ran/TC4, and NTF2. NLS receptors appear to function as shuttling carriers that transport NLS-containing substrates from the cytoplasm to the nuclear interior. During trasnport through the NPC, the complex formed by the NLS receptor and substrate is transferred in a stepwise fashion between a number of discrete NPC components, including peripheral and internal binding sites, and a central gated channel. We are beginning to elucidate the functions of Ran and NTF2 in this process, and have characaterized specific NPC proteins that interact with these factors.
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.
In mammalian somatic cells, the nuclear lamina contains mainly a polymer of two to four intermediate filament proteins called lamins. To more clearly understand its functions as a nuclear scaffold, we have been characterizing the interactions of the lamina with the inner nuclear membrane and chromatin. We have identified two integral membrane proteins of the nuclear envelope, LAP1 and LAP2, that are likely to be involved in the attachment of the lamina to the membrane. Each protein binds to distinctive groups of lamins. LAP2 also specifically binds to chromatin and probably contributes to the interaction between chromatin and the nuclear envelope. Using cDNA cloning, we found that LAP1 and LAP2 have single membrane-spanning segments and large nucleoplasmic domains, consistent with their putative roles as membrane anchors.
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.