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The Ran GTPase pathway
Ran is a small GTPase that is required for nuclear transport, cell cycle control, mitotic spindle formation and post-mitotic nuclear assembly. Ran is regulated by a cytosolic GTPase activating protein, RanGAP1, and by a chromatin-bound nucleotide exchange factor, RCC1. The distribution of Ran-GTP provides important spatial information that directs cellular activities during different parts of the cell cycle. During interphase, the localization of factors for Ran's nucleotide exchange and hydrolysis strongly predicts that nuclear Ran is GTP-bound and cytosolic Ran is GDP-bound. This compartmentalization determines the directionality of nuclear transport by promoting the loading and unloading of transport receptors in a manner that is appropriate to the nucleus or cytosol.
In mitosis, microtubules (MTs) are stabilized in the vicinity of chromatin by a factor(s) whose concentration is inversely proportional to distance from chromosomes. This stabilization contributes toward the appropriate assembly of bipolar mitotic spindles. Ran was recently implicated in spindle assembly through observations in M-phase Xenopus egg extracts, including the finding that elevated levels of Ran-GTP promote spontaneous MT polymerization in a manner that is independent of chromosomes. Since a significant fraction of RCC1 remains chromatin-associated in mitosis, it has been widely hypothesized that Ran-GTP could be a diffusionally-distributed stabilization factor that contributes to the localized stabilization of MTs near condensed chromosomes.
This model of Ran function obviously predicts that the Ran pathway is dramatically re-arranged as cells enter and exit mitosis, and that these re-arrangements are carefully regulated. We are currently investigating the behavior of a subset of the core regulators of Ran (RanGAP1, RanBP1, RanBP2 and RCC1) during mitosis. We are particularly interested in the spatial regulation of RanGAP1 through modification by SUMO-1.
Figure 1: The Ran pathway.
During interphase, Ran promotes protein trafficking through the association and dissociation of transport complexes (upper panel). Complexes of import cargo and their cognate receptors form in the cytosol and translocate across the nuclear pore. In the nucleus, Ran-GTP (blue diamonds) binds to the import receptors and dissociates transport complexes. The import receptors return to the cytosol in association with Ran-GTP, where RanGAP1 and RanBP1 hydrolyze Ran-GTP to Ran-GDP, permitting recycling of the receptor. Export receptors and Ran-GTP bind to their cargo in the nucleus, and this complex translocates to the cytosol, where it is dissociated by the action of RanGAP1 and RanBP1. The compartmentalization of RanGEF, RanGAP1 and RanBP1 serve to maintain the asymmetric distribution of Ran-GTP across the nuclear envelope. The localization of RCC1 on chromatin may promote assembly in mitosis (lower panel). Importin a and importin b are thought to inhibit spindle assembly activators (SA) under low Ran-GTP at a distance from chromosomes. In the vicinity of chromosomes, increased concentration and/or activity of RCC1 may provide increased levels of Ran-GTP. Increased levels of Ran-GTP could bind to importin b, thus promote dissociation of these inhibitor complexes and allowing full activation of SAA.
The SUMO-1 protein modification pathway
SUMO-1 is a small ubiquitin-like protein. SUMO-1 can be covalently conjugated to other proteins through an isopeptide linkage in a manner similar to ubiquitin. The SUMO-1 conjugation pathway utilizes enzymes that both show sequence similarity to enzymes in the ubiquitin pathway and utilize similar biochemical mechanisms. A large and growing number of SUMO-1 conjugation substrates have been reported in vertebrates to date. Notably, the profile of SUMO-1 conjugated changes substantially in response to altered cellular conditions, suggesting that there are mechanisms to control the specificity of conjugation and/or deconjugation of SUMO-1 differentially between distinct substrates.
RanGAP1 was the first documented substrate for conjugation with SUMO-1. However, the functional significance of this conjugation has not been fully clarified. We have been particularly interested in understanding the role or SUMO-1 in regulating RanGAP1, both because of our interest in the Ran pathway and because we believe that understanding this interaction will serve as a paradigm for other SUMO-1 conjugation targets. We have recently found that SUMO-1 conjugation is required for mitotic localization of RanGAP1. Our findings suggests that a major role of SUMO-1 conjugation to RanGAP1 may be the spatial regulation of the Ran pathway during mitosis. Together, these results imply that Ran-GTP gradients may be regulated in mitosis in a manner that is significantly more complex than previously anticipated.
In order to study the SUMO-1 pathway more generally, we have also been examining the enzymes responsible for its conjugation to and deconjugation from other cellular proteins. We are currently undertaking a strategy to directly disrupt these proteins and examine the consequences for the Ran pathway and other cellular functions.
Figure 2: The SUMO-1 conjugation pathway.
SUMO-1 is initially proteolytically processed by C-terminal hydrolases to its active form. It then serves as the substrate in the ATP-dependent formation of an isopeptide bond between the free carboxyl group of the C-terminal glycine in SUMO and the e-amino group of a lysine in the acceptor protein. This reaction is mediated by Aos1/Uba2 (E1 enzyme) and Ubc9 (E2 enzyme). E3 ligases have recently been described for this system, but little is known regarding their mechanism and function. Cleavage of the isopeptide bond is mediated by isopeptidases.