The small GTP binding protein Ran is an essential component of the nuclear protein import machinery whose GTPase cycle is regulated by the nuclear guanosine nucleotide exchange factor RCC1 and by the cytosolic GTPase activating protein RanGAP. In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae the RanGAP activity is encoded by the RNA1 genes which are essential for cell viability and nucleocytoplasmic transport in vivo. Although of limited sequence identity the two yeast proteins show a conserved structural organization characterized by an N-terminal domain of eight leucine-rich repeats, motifs implicated in protein-protein interactions, and a C-terminal domain rich in acidic amino acid residues. By analyzing the RanGAP activity of a series of recombinantly expressed rna1p mutant derivatives, we show that the highly acidic sequence in the C-terminal domain of both yeast proteins is indispensable for activating Ran-mediated GTP hydrolysis. Chemical cross-linking reveals that the same sequence in rna1p is required for rna1p⅐Ran complex formation indicating that the loss of GAP activity in the C-terminally truncated rna1p mutants results from an impaired interaction with Ran. The predominant species stabilized through the covalent cross-link is a rna1p⅐Ran heterodimer whose formation requires the GTP-bound conformation of Ran. As the acidic C-terminal domain of rna1p is required for establishing the interaction with Ran, the leucine-rich repeats domain in rna1p is potentially available for additional protein interactions perhaps required for directing a fraction of rna1p to the nuclear pore.The import of karyophilic proteins into the nucleus which occurs through the nuclear pore complex (NPC) 1 is an energydependent process specified by nuclear localization signals (NLSs) on the import substrate (for reviews see Refs. 1-4). Using digitonin-permeabilized HeLa cells as an in vitro transport system, it was shown that the import of substrate proteins is a multistep process depending on four cytosolic factors (5, 6) (for reviews see Refs. 7-10). Two of these factors form a heterodimeric complex serving as the NLS receptor. The smaller 60-kDa subunit of this complex, also known as importin ␣, is responsible for NLS binding, whereas the larger 97-kDa subunit, importin ␤, most likely mediates a targeting of the substrate-NLS receptor complex to the NPC. After docking the entire substrate-receptor complex is thought to travel through the NPC and then to dissociate close to or at the nucleoplasmic side of the pore (for review see Ref. 11). The two other factors identified in the in vitro transport system as essential components of the nuclear import machinery are the GTP-binding protein Ran/TC4 (herein referred to as Ran (12, 13)) and a small protein of 10 kDa, p10/NTF2. The latter can interact with NPC proteins, Ran, and importin ␤ and appears to be involved in the formation of a pentameric complex including p10, Ran in its GDP-bound form, the two NLS receptor subunits and a nuclear pore protein (14, 15).R...

GTPase activating proteins (GAPs) for Ran, a Ras-related GTPase participating in nucleocytoplasmic transport, have been identified in different species ranging from yeast to man. All RanGAPs are characterized by a conserved domain consisting of eight leucine-rich repeats (LRRs) interrupted at two positions by so-called separating regions, the latter being unique for RanGAPs within the family of LRR proteins. The cytosolic RanGAP activity is essential for the Ran GTPase cycle which in turn provides directionality in nucleocytoplasmic transport, but the structural basis for the interaction between Ran and its GAP has not been elucidated. In order to gain a better understanding of this interaction we generated a number of mutant RanGAPs carrying amino acid substitutions in the LRR domain and analysed their complex formation with Ran as well as their ability to stimulate the intrinsic GTPase activity of the G protein. We show that conserved charged residues present in the separating regions of the LRR domain are indispensable for efficient Ran binding and GAP activity. These separating regions contain three conserved arginines which could possibly serve as catalytic residues similar to the arginine fingers identified in GAPs for other small GTPases. However, mutations in two of these arginines do not affect the GAP activity and replacement of the third conserved arginine (Arg91 in human RanGAP) severely interferes not only with GAP activity but also with Ran binding. This indicates that RanGAP-stimulated GTP hydrolysis on Ran does not involve a catalytic arginine residue but requires certain charged residues of the LRR domain of the GAP for mediating the protein-protein interaction.

Disease progression and clinical diagnostics of a number of hereditable metabolic diseases are determined by organ involvement in disturbed deposition of certain molecules. Current clinical imaging is unable to visualize this maldistribution with sufficient specificity and sensitivity, such as in Wilson's disease. The quest for understanding cellular Cu distribution in these patients requires element- and molecule-specific images with nanometer-scale spatial resolution. We have used a new cryo-mass spectrometric instrument with an integrated cryosectioning chamber for preparation and analysis of frozen hydrated samples of Wilson's disease tissue. With laser post-ionization secondary neutral mass spectrometry (laser-SNMS), we were able to image Cu and other intrinsic elements and molecules in less than 1 mg of frozen hydrated liver tissue from a murine model of Wilson's disease. A 40-50 times higher Cu concentration was measured in the disease tissue as compared to the control mouse. Furthermore, major histomorphological changes were observed using this advanced nano-science tool. The results showed that the combination of in-vacuum cryosectioning and cryo-laser-SNMS technologies is particularly well suited for identifying specific cell structures and imaging trace element concentrations with subcellular resolution and upper-parts-per-billion sensitivity in biological samples. This technology can provide a novel diagnostic tool for clinical applications in various diseases involving trace elements.

GTPase activating proteins (GAPs) for Ran, a Ras-related GTPase participating in nucleocytoplasmic transport, have been identified in different species ranging from yeast to man. All RanGAPs are characterized by a conserved domain consisting of eight leucine-rich repeats (LRRs) interrupted at two positions by so-called separating regions, the latter being unique for RanGAPs within the family of LRR proteins. The cytosolic RanGAP activity is essential for the Ran GTPase cycle which in turn provides directionality in nucleocytoplasmic transport, but the structural basis for the interaction between Ran and its GAP has not been elucidated. In order to gain a better understanding of this interaction we generated a number of mutant RanGAPs carrying amino acid substitutions in the LRR domain and analysed their complex formation with Ran as well as their ability to stimulate the intrinsic GTPase activity of the G protein. We show that conserved charged residues present in the separating regions of the LRR domain are indispensable for efficient Ran binding and GAP activity. These separating regions contain three conserved arginines which could possibly serve as catalytic residues similar to the arginine fingers identified in GAPs for other small GTPases. However, mutations in two of these arginines do not affect the GAP activity and replacement of the third conserved arginine (Arg91 in human RanGAP) severely interferes not only with GAP activity but also with Ran binding. This indicates that RanGAP-stimulated GTP hydrolysis on Ran does not involve a catalytic arginine residue but requires certain charged residues of the LRR domain of the GAP for mediating the protein-protein interaction.

The amino acid sequence of Rnalp, a yeast protein implicated in the maturation and/or nucleocytoplasmic transport of RNA, is characterised by the presence of eight leucine-rich repeats (LLRs) as well as two intervening repeats of a different type and a highly acidic C-terminal region. Limited proteolysis of purified Rnalp expressed recombinantly in bacteria reveals that the C-terminal extension but not the region containing the two types of repeats is highly accessible to proteolytic attack and that the C-terminal region most likely harbours (a) low affinity Ca2+-binding site(s). These results are indicative of the domain structure of the Rnalp molecule, with the repeats and the Cterminal region being accessible for different interactions.