Larry Gerace

A Small Ubiquitin-Related Polypeptide Involved in Targeting RanGAP1 to Nuclear Pore Complex Protein RanBP2

We have found that the mammalian Ran GTPase-activating protein RanGAP1 is highly concentrated at the cytoplasmic periphery of the nuclear pore complex (NPC), where it associates with the 358-kDa Ran-GTP-binding protein RanBP2. This interaction requires the ATP-dependent posttranslational conjugation of RanGAP1 with SUMO-1 (for small ubiquitin-related modifier), a novel protein of 101 amino acids that contains low but significant homology to ubiquitin. SUMO-1 appears to represent the prototype for a novel family of ubiquitin-related protein modifiers. Inhibition of nuclear protein import resulting from antibodies directed at NPC-associated RanGAP1 cannot be overcome by soluble cytosolic RanGAP1, indicating that GTP hydrolysis by Ran at RanBP2 is required for nuclear protein import.

The nuclear lamina is a meshwork of intermediate-type filaments

The nuclear lamina, a protein meshwork lining the nucleoplasmic surface of the inner nuclear membrane1,2, is thought to provide a framework for organizing nuclear envelope structure3 and an anchoring site at the nuclear periphery for interphase chromatin3–5. In several higher eukaryotic cells, the lamina appears to be a polymer comprised mainly of one to three immunologically related polypeptides of relative molecular mass (Mr) 60,000–75,000 (60–70K) termed lamins1,2. Three lamins (A, B, and C) are typically present in mammalian somatic cells. Previous studies on nuclear envelopes of rat liver6 and Xenopus oocytes7 suggested that the lamina has a fibrillar or filamentous substructure. Interestingly, protein sequences recently deduced for human lamins A and C from complementary DNA clones8,9 indicate that both of these polypeptides contain a region of ∼350 amino acids very similar in sequence to the coiled-coil α-helical rod domain that characterizes all intermediate-type filament (IF) proteins10,11. Here we analyse the supramolecular organization of the native nuclear lamina and the structure and assembly properties of purified lamins, and show that the lamins constitute a previously unrecognized class of IF polypeptides.

Integral membrane proteins of the nuclear envelope interact with lamins and chromosomes, and binding is modulated by mitotic phosphorylation

Lamina-associated polypeptides (LAPs) 1A, 1B, 1C, and 2 are integral membrane proteins of the nuclear envelope associated with the nuclear lamina. Using in vitro assays, we show that LAPs 1A and 1B specifically bind to both lamins A and C and lamin B1, while LAP 2 associates only with lamin B1. LAP 2 also binds to mitotic chromosomes. The LAPs are phosphorylated during mitosis, and phosphorylation of LAP 2 by mitotic cytosol inhibits its binding to both lamin B1 and chromosomes. During late anaphase, LAP 2 associates with chromosomes prior to assembly of most lamins. Together, these data suggest that LAP 2 may have a key role in initial events of nuclear envelope reassembly, and that both LAP 2 and LAP 1 may be involved in attaching lamins to the nuclear envelope.

A cell free system to study reassembly of the nuclear envelope at the end of mitosis

We described a cell free system involving total homogenates of metaphase CHO cells, which yields telophase-like assembly of nuclear envelopes around mitotic chromosomes. During formation of the nuclear envelope in vitro, the three major lamina polypeptides (lamins A, B, and C) assemble around chromosomes and become dephosphorylated, similar to their behavior in vivo during telophase. Nuclear lamina and envelope assembly apparently do not require free ATP and are strongly inhibited by gamma-S-ATP, supporting the notion that these processes are regulated by protein dephosphorylation. Immunological depletion of disassembled lamins from the initial assembly system results in strong inhibition of subsequent nuclear envelope assembly, directly demonstrating that the lamins are involved in this process.

Adenovirus protein VI mediates membrane disruption following capsid disassembly.

ABSTRACT In contrast to enveloped viruses, the mechanisms involved in membrane penetration by nonenveloped viruses are not as well understood. In these studies, we determined the relationship between adenovirus (Ad) capsid disassembly and the development of membrane lytic activity. Exposure to low pH or heating induced conformational changes in wild-type Ad but not in temperature-sensitive Ad (ts1) particles that fail to escape the early endosome. Wild-type Ad but not ts1 particles permeabilized model membranes (liposomes) and facilitated the cytosolic delivery of a ribotoxin. Alterations in wild-type Ad capsids were associated with the exposure of a pH-independent membrane lytic factor. Unexpectedly, this factor was identified as protein VI, a 22-kDa cement protein located beneath the peripentonal hexons in the viral capsid. Recombinant protein VI and preprotein VI, but not a deletion mutant lacking an N-terminal amphipathic α-helix, possessed membrane lytic activity similar to partially disassembled virions. A new model of Ad entry is proposed based on our present observations of capsid disassembly and membrane penetration.

Mechanisms of nuclear protein import

The past two years have seen a significant increase in our understanding of nuclear protein import. Five cytosolic import factors have been identified, two of which have been shown to directly interact with components of the nuclear pore complex. These findings enable refinement of previous models for steps in the nuclear import pathway, and provide a framework for future research.

Nuclear export signals and the fast track to the cytoplasm.

One of the defining features of eukaryotic cells is the segregation of RNA biogenesis and DNA replication in the nucleus, separate from the cytoplasmic machinery for protein synthesis. Integration of the activities of the nucleus and cytoplasm requires the continuous transport of proteins, RNAs, and small molecules between these two compartments. Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs), large supramolecular structures that span the nuclear envelope (reviewed by Rout and Wente, 1994). NPCs contain aqueous channels with a diameter of 10 nm, which allow ions, metabolites, and small proteins to diffuse passively between the nucleus and cytoplasm. Most proteins and RNAs are too large to cross the NPC by passive diffusion at physiologically relevant rates. Instead, they are transported through a gated transport channel in the NPC by active mechanisms, which are saturable and energy dependent and involve specific signals on the transported molecules (reviewed by Izaurralde and Mattaj, 1995; Melchior and Gerace, 1995). While substantial insight into the molecular basis for nuclear protein import has been obtained recently, nuclear export remains poorly understood. However, four papers in this issue of Cell (Wen et al., 1995; Fischer et al., 1995; Bogerd et al., 1995; Stutz et al., 1995) characterize molecular signals and their interacting components involved in the nuclear export of protein and RNA and herald a new period in the study of nuclear export mechanisms. Molecular Signals for Nuclear Import and Export A watershed for the analysis of nuclear protein import was the discovery that short amino acid stretches termed nuclear localization signals (N LSs) specify the nuclear import of many proteins. While N LSs lack a strict consensus, they are usually highly enriched in basic amino acids (reviewed by Dingwall and Laskey, 1991). NLSs containing both single and bipartite stretches of basic residues have been described (Figure 1), These N LSs can function when transplanted to nonnuclear proteins, either by inserting NLSspecifying codons into cDNAs or by coupling synthetic peptides containing NLSs to folded proteins. Characterization of nuclear export signals (NESs) has lagged considerably behind analysis of NLSs. This partly is due to the experimental difficulty of studying nuclear export, but also relates to the complex nature of many of the molecular species that are exported from the nucleus (discussed by Izaurralde and Mattaj, 1995). The RNA substrates for nuclear export are probably all transported as RNA-protein (RNP) complexes. Many or all of these RNPs contain multiple polypeptides, so it is difficult to know the precise composition of the substrate that is actually transMinireview

Organization and modulation of nuclear lamina structure.

SUMMARY The nuclear lamina is a protein meshwork associated with the nucleoplasmic surface of the inner nuclear membrane, that is suggested to be important for organizing nuclear envelope and interphase chromosome architecture. To investigate the structural organization of the lamina, we have analysed rat liver nuclear envelopes by various chemical extraction procedures. From these studies, we have defined conditions that yield a nuclear envelope subtraction that is both highly enriched in the lamina and devoid of pore complexes. This fraction contains mostly lamins A, B and C, the three major lamina polypeptides that are apparently arranged in a polymeric assembly. Our chemical extraction studies also indicate that lamin B has a stronger interaction with nuclear membranes than the other two lamins, and support the possibility that lamin B is important for attaching the lamina to the inner nuclear membrane. We have examined the synthesis and assembly of the lamins during interphase in tissue-culture cells to investigate lamina structure by a second approach. We found that all three lamins are synthesized at similar rates throughout the cell cycle in synchronized Chinese hamster ovary cells, and that their biosynthesis is not temporally coupled to DNA replication. Our studies indicate that newly synthesized lamins are rapidly assembled into an insoluble lamina structure but that the apparent half-time for lamina insertion differs for individual lamins. We have also observed that lamin A is synthesized as an apparent precursor molecule that is converted to mature lamin A only after integration into the lamina structure. The lamina is reversibly depolymerized during cell division, a process that may be mediated by enzymic phosphorylation of the lamins. To investigate this possibility further, we have analysed charge-altering modifications of the lamins on two-dimensional gels, and have found that phosphorylation is the only detectable modification of these proteins that occurs specifically during mitosis. Furthermore, we have determined that when the lamins are disassembled during metaphase, each lamin has approximately 2 moles of associated phosphate/mole lamin, a value that is four to sevenfold higher than the average interphase level. Considering this information, we discuss a model by which depolymerization and reassembly of the lamina can regulate the reversible disassembly of the nuclear envelope during mitosis.

A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide with a large lumenal domain and a small cytoplasmic tail.

One of a small number of polypeptides of the nuclear pore complex that have been identified is a major glycoprotein called gp210. Since it is very resistant to chemical extractions from membranes, gp210 was suggested to be integrated into nuclear membranes. In this study we have determined the membrane topology of this protein by biochemical and immunological approaches. We found that limited proteolysis of isolated nuclear envelopes with papain released a 200 kd water‐soluble fragment of gp210 containing concanavalin A‐reactive carbohydrate. Immunogold electron microscopy with a monoclonal antibody showed that this domain is localized on the lumenal side of nuclear membranes at pore complexes. Anti‐peptide antibodies against two sequences near the C‐terminus of gp210 were used to map possible membrane spanning and cytoplasmically disposed regions of this protein. From analysis of the protease sensitivity of these epitopes in sealed membrane vesicles, we determined that gp210 contains a small cytoplasmic tail and only a single membrane‐spanning region. Thus, gp210 is a transmembrane protein with most of its mass, including the carbohydrate, located in the perinuclear space. This topology suggests that gp210 is involved primarily in structural organization of the pore complex, for which it may provide a membrane attachment site.

Cloning of a cDNA for lamina-associated polypeptide 2 (LAP2) and identification of regions that specify targeting to the nuclear envelope.

Lamina‐associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane, which binds directly to both lamin B1 and chromosomes in a mitotic phosphorylation‐regulated manner. The biochemical and physiological properties of LAP2 suggest an important role in nuclear envelope re‐assembly at the end of mitosis and/or anchoring of the nuclear lamina and interphase chromosomes to the nuclear envelope. We describe the cDNA cloning of LAP2 and characterization of its membrane topology and targeting to the nuclear envelope. The LAP2 cDNA sequence predicts a protein of 452 amino acids, containing a large hydrophilic domain with several potential cdc2 kinase phosphorylation sites and a single putative membrane‐spanning sequence at residues 410‐433. Immunogold localization of an LAP2 epitope in isolated nuclear envelopes indicates that the large amino‐terminal hydrophilic domain (residues 1‐409) is exposed to the nucleoplasm. By expressing deletion mutants of LAP2 in cultured cells, we have identified multiple regions in its nucleoplasmic domain that promote localization at the nuclear envelope. These data suggest that targeting of LAP2 to the nuclear envelope is mediated by cooperative interactions with multiple binding sites at the inner nuclear membrane.