302 There have been three major advances in the understanding of the Ran pathway during the past two years: first, a general model for Ran’s function in nuclear transport has been proposed and extensively tested. Second, crystal structures for many proteins that regulate or interact with Ran have been reported, which provide molecular details of how Ran works. Third, it has been documented that Ran regulates mitotic spindle assembly in a transport-independent fashion. Addresses Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, National Institutes of Health, Building 18, Room 106, Bethesda, MD 20892-5431, USA *e-mail: mdasso@helix.nih.gov Current Opinion in Cell Biology 2000, 12:302-307 0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations BHK baby hamster kidney NTF2 nuclear transport factor 2 PCC premature chromosome condensation RBD1 Ran-binding domain 1 RanBP1 Ran-binding protein 1 RanGAP Ran GTPase activating protein RanGEF Ran guanine nucleotide exchange factor Introduction One of the defining features of eukaryotic cells is the com- partmentalization of DNA within the nucleus. In order to maintain the nucleus as a distinct cellular compartment, it is necessary to define its molecular identity and regulate the trafficking of macromolecules to and from it. During the past five years, the small GTPase Ran has emerged as an essential player in nuclear transport [1]. It both defines compartmental identity and specifies directionality of nuclearcytoplasmic transport. It has also become clear that Ran is essential for other cellular activities, such as nuclear assembly, mitotic cell-cycle regulation and spindle assem- bly. In this review, we discuss the roles of Ran in different nuclear processes and how these roles may be related to each other. Due to the brevity of this review, we empha- size recent advances in our understanding of these processes in metazoans. We therefore apologize to our col- leagues whose work on yeast cannot be discussed here. Fundamentals of the Ran pathway Ran is an extremely abundant and soluble GTPase located predominantly within the nucleus of interphase metazoan cells [2]. Like other Ras family members, Ran’s associations with different cellular proteins are dependent upon the nucleotide that is bound to it. Regulatory proteins increase the rates of nucleotide exchange and nucleotide hydrolysis, allowing Ran to cycle between the GTP- and GDP-bound states at physiological rates (Figure 1). A guanine nucleotide exchange factor (RanGEF) promotes release of guanine nucleotides, allowing Ran to acquire GTP. RanGEF is local- ized within nuclei throughout interphase and is bound to chromatin [3]. A GTPase activating protein (RanGAP) stim- ulates nucleotide hydrolysis. RanGAP is localized in the cytosol during interphase [4,5]. The localization of Ran’s regulators would suggest that Ran is primarily GTP-bound in the nucleus and GDP-bound in the cytoplasm. It would also suggest that Ran moves between these compartments in order to undergo a complete round of GTP binding and hydrolysis. Maintenance of Ran’s subcellular localization also requires a small Ran-GDP binding protein called nuclear transport factor 2 (NTF2) [6 •• ,7]. Three other classes of proteins bind directly to Ran, in addition to the regulators discussed above. The first class is a group of Ran-GTP-binding proteins with homology to Ran-binding protein 1 (RanBP1) [8]. These proteins do not have RanGAP activity, but they can increase the rate of RanGAP-mediated Ran-GTP hydrolysis in vitro approxi- mately ten-fold [9]. A second class includes importin-β and related transport receptors. These proteins bind to Ran- GTP in a manner that promotes their association to, or dissociation from, their transport substrates after entry into or exit from the nucleus. Thirdly, recent reports have shown that Ran-GDP can associate with zinc finger domains of nuclear pore proteins [10,11]. The crystal structures of Ran and a number of its associat- ed proteins have been solved and have revealed important molecular details about this pathway. The switch I and II The role of Ran in nuclear function Yoshiaki Azuma and Mary Dasso* Figure 1 Enzymes regulating the Ran GTPase cycle. RanGEF, a chromatin- bound nucleotide exchange factor, catalyzes guanine nucleotide exchange on Ran. RanGAP, a cytosolic GTPase activating protein, catalyzes GTP-hydrolysis in association with RanBP1, an accessory protein. Both of these reactions are accelerated five to six orders of magnitude by RanGAP and RanGEF in vitro. In vivo, exchange and hydrolysis are compartmentalized to the nucleus and cytoplasm respectively, which is predicted to produce a steep gradient of Ran- GTP across the nuclear envelope. For more details, see text. GTP GDP P i RanGAP RanBP1 RanGEF Ran- GTP Ran- GDP Nucleus Cytoplasm Current Opinion in Cell Biology