Caspases target only two architectural components within the core structure of the nuclear pore complex

Caspases were recently implicated in the functional impairment of the nuclear pore complex during apoptosis, affecting its dual activity as nucleocytoplasmic transport channel and permeability barrier. Concurrently, electron microscopic data indicated that nuclear pore morphology is not overtly altered in apoptotic cells, raising the question of how caspases may deactivate nuclear pore function while leaving its overall structure largely intact. To clarify this issue we have analyzed the fate of all known nuclear pore proteins during apoptotic cell death. Our results show that only two of more than 20 nuclear pore core structure components, namely Nup93 and Nup96, are caspase targets. Both proteins are cleaved near their N terminus, disrupting the domains required for interaction with other nucleoporins actively involved in transport and providing the permeability barrier but dispensable for maintaining the nuclear pore scaffold. Caspase-mediated proteolysis of only few nuclear pore complex components may exemplify a general strategy of apoptotic cells to efficiently disable huge macromolecular machines.     

Should I stay or should I go? Nucleocytoplasmic trafficking in plant innate immunity

Communication between the cytoplasm and the nucleus is a fundamental feature of eukaryotic cells. Bidirectional transport of macromolecules across the nuclear envelope is typically mediated by receptors and occurs exclusively through nuclear pore complexes (NPCs). The components and molecular mechanisms regulating nucleocytoplasmic trafficking and signalling processes are well studied in animals and yeast but are poorly understood in plants. Current work shows that components of the NPC and the nuclear import and export machinery play essential roles in plant innate immunity. Translocation of defence regulators and Resistance (R) proteins between the cytoplasm and the nucleus are recently uncovered aspects of plant defence responses against pathogens. Future studies will reveal more details on the spatial and temporal dynamics and regulation of this process.     

The nuclear pore complex

The nuclear pore complex is the largest supramolecular complex that assembles in the eukaryotic cell. This structure is highly dynamic and must disassemble prior to mitosis and reassemble after the event. The directed movement of macromolecules into and out of the nucleus occurs through the nuclear pore complex, a potentially regulatory point for translocation. Using biochemical and genetic approaches, several nuclear pore complex proteins from yeast and vertebrates have been well characterized. Although very little is known about plant nuclear pore proteins, research is providing new information that indicates that plant nuclear pore complexes may have some unique features.     

GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import

Mediated import of proteins into the nucleus involves multiple cytosolic factors, including the small GTPase Ran. Whether Ran functions by interacting with other cytosolic proteins or components of the nuclear pore complex has been unclear. Furthermore, the precise transport step where Ran acts has not been determined. To address these questions, we have analyzed the binding interactions of Ran using permeabilized cells and isolated nuclear envelopes. By light and electron microscope immunolocalization, we have found that Ran accumulates specifically at the cytoplasmic surface of the nuclear pore complex when nuclear import in permeabilized cells is inhibited by nonhydrolyzable analogs of GTP. Ran associates with a peripheral pore complex region that is similar to the area where transport ligands accumulate by depletion of ATP, which arrests an early step of transport. Binding studies with isolated nuclear envelopes in the absence of added cytosol indicate that Ran-GTP directly interacts with a pore complex protein. Using blot overlay techniques, we detected a single prominent polypeptide of isolated nuclear envelopes that binds Ran-GTP. This corresponds to the 358-kD protein RanBP2, a Ran binding pore complex protein recently identified by two-hybrid screening. Thus, RanBP2 is likely to constitute the Ran-GTP-binding site detected at the cytoplasmic periphery of the pore complex. These data support a model in which initial ligand binding to the nuclear pore complex occurs at or near RanBP2, and that hydrolysis of GTP by Ran at this site serves to define commitment to the nuclear import pathway.     

Nuclear transport and nuclear pores in yeast

The central features of nuclear import have been conserved during evolution. In yeast the nuclear accumulation of proteins follows the same selective and active transport mechanisms known from higher eukaryotes. Yeast nuclear proteins contain nuclear localization sequences (NLS) which are presumably recognized by receptors in the cytoplasm and the nuclear envelope. Subsequent to this recognition step, nuclear proteins are translocated into the nucleus via the nuclear pore complexes. The structure of the yeast nuclear pore complex resembles that of higher eukaryotes. Recently, the first putative components of the yeast nuclear import machinery have been cloned and sequenced. The genetically amenable yeast system allows for an efficient structural and functional analysis of these components. Due to the evolutionary conservation potential insights into the nuclear import mechanisms in yeast can be transferred to higher eukaryotes. Thus, yeast can be considered as a eukaryotic model system to study nuclear transport.     

NUA Activities at the Plant Nuclear Pore

NUA (Nuclear Pore Anchor), the Arabidopsis homolog of Tpr (Translocated Promoter Region), is one of the few nuclear pore proteins conserved between animals, yeast and plants. In the May issue of Plant Cell, we report that null mutants of NUA show a pleiotropic, early flowering phenotype accompanied by changes in SUMo and RNA homeostasis. We have shown that the early flowering phenotype is caused by changed abundances of flowering time regulators involved in several pathways. Arabidopsis nua mutants phenocopy mutants lacking the ESD4 (EARlY IN ShoRT DAYS 4) SUMo protease, similar to mutants of their respective yeast homologs. however, in contrast to the comparable yeast mutants, ESD4 does not appear to be delocalized from the nuclear pore in nua mutants. Taken together, our experimental data suggests a role for NUA in controlling mRNA export from the nucleus as well as SUMo protease activity at the nuclear pore, comparable but not identical to its homologs in other eukaryotes. Furthermore, characterization of NUA illustrates a potential link at the nuclear pore between SUMo modification, RNA homeostasis and plant developmental control.Key Words: nuclear pore complex, nucleoporin, nuclear envelope, nucleocytoplasmic transport, SUMO, mRNA export, flowering time     

Transport pathways of macromolecules between the nucleus and the cytoplasm.

Transport between the nucleus and cytoplasm involves both stationary components and mobile factors acting in concert to move macromolecules through the nuclear pore complex. Multiple transport pathways requiring both unique and shared components have been identified. In the past 18 months, new findings have shed light on the nature of some of the mobile components of these pathways. New receptor-cargo pairs for both import and export pathways have been identified extending the breadth of known transport pathways. Surprising findings on the role of Ran and energy in transport have changed our way of thinking about the mechanism of movement through the nuclear pore.     

Nuclear Pore Complex Structure in Birds

The nuclear envelope consists of two parallel membranes enclosing an aqueous lumen. In places there are pores in both membranes at which the two membranes are joined. Within these pores reside the nuclear pore complexes. The current structural models of the nuclear pore complex have been derived from a number of studies using different electron microscopical techniques. Recently, using surface imaging techniques such as field emission in-lens scanning electron microscopy, novel structures have been identified, particularly at the periphery of the structure, most notably the nucleoplasmic basket. One limitation of the current models is that they are based almost entirely on nuclear envelopes isolated from amphibian oocytes and a pressing question is whether this structure is the same in other organisms and tissues. Here we have studied the structure of nuclear envelopes isolated from bird oocytes. We show that the overall structure is remarkably conserved. In particular, recently discovered peripheral structures appear very similar. We see variations in basket conformation but believe that this is related to the functional states of individual pore complexes.     

Nucleocytoplasmic transport of macromolecules.

Nucleocytoplasmic transport is a complex process that consists of the movement of numerous macromolecules back and forth across the nuclear envelope. All macromolecules that move in and out of the nucleus do so via nuclear pore complexes that form large proteinaceous channels in the nuclear envelope. In addition to nuclear pores, nuclear transport of macromolecules requires a number of soluble factors that are found both in the cytoplasm and in the nucleus. A combination of biochemical, genetic, and cell biological approaches have been used to identify and characterize the various components of the nuclear transport machinery. Recent studies have shown that both import to and export from the nucleus are mediated by signals found within the transport substrates. Several studies have demonstrated that these signals are recognized by soluble factors that target these substrates to the nuclear pore. Once substrates have been directed to the pore, most transport events depend on a cycle of GTP hydrolysis mediated by the small Ras-like GTPase, Ran, as well as other proteins that regulate the guanine nucleotide-bound state of Ran. Many of the essential factors have been identified, and the challenge that remains is to determine the exact mechanism by which transport occurs. This review attempts to present an integrated view of our current understanding of nuclear transport while highlighting the contributions that have been made through studies with genetic organisms such as the budding yeast, Saccharomyces cerevisiae.     

Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle

Macromolecular transport between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC) and is mediated by multiple families of soluble transport factors. All these transport factors share the ability to translocate across the NPC through specific interactions with components of the nuclear pore. This review highlights advances in our understanding of the structure and function of the NPC and the shuttling transport receptors involved in nuclear transport. It discusses recently proposed models for the translocation of receptor-cargo complexes through the NPC channel and reviews how the small GTPase Ran functions as a positional marker of the genome to regulate multiple important aspects of the eukaryotic cell cycle.     

Reactive oxygen species as signals that modulate plant stress responses and programmed cell death

Reactive oxygen species (ROS) are known as toxic metabolic products in plants and other aerobic organisms. An elaborate and highly redundant plant ROS network, composed of antioxidant enzymes, antioxidants and ROS-producing enzymes, is responsible for maintaining ROS levels under tight control. This allows ROS to serve as signaling molecules that coordinate an astonishing range of diverse plant processes. The specificity of the biological response to ROS depends on the chemical identity of ROS, intensity of the signal, sites of production, plant developmental stage, previous stresses encountered and interactions with other signaling molecules such as nitric oxide, lipid messengers and plant hormones. Although many components of the ROS signaling network have recently been identified, the challenge remains to understand how ROS-derived signals are integrated to eventually regulate such biological processes as plant growth, development, stress adaptation and programmed cell death.     

Deletion of core components of the plastid protein import machinery causes differential arrest of embryo development in Arabidopsis thaliana

Among the genes that have recently been pinpointed to be essential for plant embryo development a large number encodes plastid proteins suggesting that embryogenesis is linked to plastid localized processes. However, nuclear encoded plastid proteins are synthesized as precursors in the cytosol and subsequently have to be transported across the plastid envelopes by a complex import machinery. We supposed that deletion of components of this machinery should allow a more general assessment of the role of plastids in embryogenesis since it will not only affect single proteins but instead inhibit the accumulation of most plastid proteins. Here we have characterized three Arabidopsis thaliana mutants lacking core components of the Toc complex, the protein translocase in the outer plastid envelope membrane, which indeed show embryo lethal phenotypes. Remarkably, embryo development in the atToc75-III mutant, lacking the pore forming component of the translocase, was arrested extremely early at the two-cell stage. In contrast, despite the complete or almost complete lack of the import receptors Toc34 and Toc159, embryo development in the a tToc33/34 and atToc132/159 mutants proceeded slowly and was arrested later at the transition to the globular and the heart stage, respectively. These data demonstrate a strict dependence of cell division and embryo development on functional plastids as well as specific functions of plastids at different stages of embryogenesis. In addition, our analysis suggest that not all components of the translocase are equally essential for plastid protein import in vivo.     

Nuclear and cytoplasmic organization in Xenopus oocytes after disruption of actin filaments by latrunculin

Actin-containing filaments have been visualized inside Xenopus oocyte nuclei by a combination of fluorescence and transmission electron microscopy. It was shown that these filaments contact nucleoli, spherical bodies, and nuclear pore complexes. The incubation of oocytes with actin-depolymerizing agent, latrunculin, caused membrane vesiculation in cytoplasm and the disruption of nucleoplasm and the integrity of the nuclear envelope. We suggest that actin-containing filaments are important cell components involved in the regulation of nucleus-cytoplasm interactions, as well as of cellular transport of components during the growth of Xenopus oocytes.     

Insights into the G1/S transition in plants

The G1/S transition generally represents the principal point of commitment to cell division. Many of the components of the cell cycle core machinery regulating the G1/S transition in plants have been recently identified. Although plant regulators of the G1/S transition display structural and biochemical homologies with their animal counterparts, their functions in integrating environmental stimuli and the developmental program within cell cycle progression are often plant-specific. In this review, recent progress in understanding the role of plant G1/S transition regulators is presented. Emerging evidence concerning the mechanisms of G1/S control in response to factors triggering the cell cycle and the integration of these mechanisms with plant development is also discussed.