RCC1 (the regulator of chromosome condensation) stimulates guanine nucleotide dissociation on the Ras‐related nuclear protein Ran. Both polypeptides are components of a regulatory pathway that has been implicated in regulating DNA replication, onset of and exit from mitosis, mRNA processing and transport, and import of proteins into the nucleus. In a search for further members of the RCC1‐Ran signal pathway, we have identified proteins of 23, 45 and 300 kDa which tightly bind to Ran‐GTP but not Ran‐GDP. The purified soluble 23 kDa Ran binding protein RanBP1 does not activate RanGTPase, but increases GTP hydrolysis induced by the RanGTPase‐activating protein RanGAP1 by an order of magnitude. In the absence of RanGAP, it strongly inhibits RCC1‐induced exchange of Ran‐bound GTP. In addition, it forms a stable complex with nucleotide‐free RCC1‐Ran. With these properties, it differs markedly from guanine diphosphate dissociation inhibitors which preferentially prevent the exchange of protein‐bound GDP and in some cases were shown to inhibit GAP‐induced GTP hydrolysis. RanBP1 is the first member of a new class of proteins regulating the binding and hydrolysis of GTP by Ras‐related proteins.

Gsp1p, the essential yeast Ran homologue, is a key regulator of transport across the nuclear pore complex (NPC). We report the identification of Yrb4p, a novel Gsp1p binding protein. The 123 kDa protein was isolated from Saccharomyces cerevisiae cells and found to be related to importin-β, the mediator of nuclear localization signal (NLS)-dependent import into the nucleus, and to Pse1p. Like importin-β, Yrb4p and Pse1p specifically bind to Gsp1p-GTP, protecting it from GTP hydrolysis and nucleotide exchange. The GTPase block of Gsp1p complexed to Yrb4p or Pse1p is released by Yrb1p, which contains a Gsp1p binding domain distinct from that of Yrb4p. This might reflect an in vivo function for Yrb1p. Cells disrupted for YRB4 are defective in nuclear import of ribosomal protein L25, but show no defect in the import of proteins containing classical NLSs. Expression of a Yrb4p mutant deficient in Gsp1p-binding is dominantlethal and blocks bidirectional traffic across the NPC in wild-type cells. L25 binds to Yrb4p and Pse1p and is released by Gsp1p-GTP. Consistent with its putative role as an import receptor for L25-like proteins, Yrb4p localizes to the cytoplasm, the nucleoplasm and the NPC.

Biological effects of high-LET (linear energy transfer) radiation have received increasing attention, particularly in the context of more efficient radiotherapy and space exploration. Efficient cell killing by high-LET radiation depends on the physical ability of accelerated particles to generate complex DNA damage, which is largely mediated by LET. However, the characteristics of DNA damage and repair upon exposure to different particles with similar LET parameters remain unexplored. We employed high-resolution confocal microscopy to examine phosphorylated histone H2AX (γH2AX)/p53-binding protein 1 (53BP1) focus streaks at the microscale level, focusing on the complexity, spatiotemporal behaviour and repair of DNA double-strand breaks generated by boron and neon ions accelerated at similar LET values (∼135 keV μm) and low energies (8 and 47 MeV per n, respectively). Cells were irradiated using sharp-angle geometry and were spatially (3D) fixed to maximize the resolution of these analyses. Both high-LET radiation types generated highly complex γH2AX/53BP1 focus clusters with a larger size, increased irregularity and slower elimination than low-LET γ-rays. Surprisingly, neon ions produced even more complex γH2AX/53BP1 focus clusters than boron ions, consistent with DSB repair kinetics. Although the exposure of cells to γ-rays and boron ions eliminated a vast majority of foci (94% and 74%, respectively) within 24 h, 45% of the foci persisted in cells irradiated with neon. Our calculations suggest that the complexity of DSB damage critically depends on (increases with) the particle track core diameter. Thus, different particles with similar LET and energy may generate different types of DNA damage, which should be considered in future research.