Modification of Ran GTPase-activating Protein by the Small Ubiquitin-related Modifier SUMO-1 Requires Ubc9, an E2-type Ubiquitin-conjugating Enzyme Homologue

Abstract

Covalent modification of the Ran GTPase-activating protein RanGAP1 with the ubiquitin-related protein SUMO-1 promotes its association with Nup358, a component of the cytoplasmic fibrils emanating from the nuclear pore complex (1, 2). In Xenopus egg extracts, Nup358 can be found in a complex with Ubc9 (3), a structural homologue of the E2-type ubiquitin-conjugating enzymes (UBCs). Here we show that a subset of the human homologue of Ubc9 (HsUbc9) colocalizes with Ran-GAP1 at the nuclear envelope. HsUbc9 forms thiolester conjugates with recombinant SUMO-1, but not with recombinant ubiquitin, indicating that it is functionally distinct from E2-type UBCs. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. These results suggest that HsUbc9 is a component of a novel enzymatic cascade that modifies RanGAP1, and possibly other substrates, with SUMO-1.The transport of selected proteins and nucleic acids across the nuclear pore complex (NPC) 1 is regulated by the small GTPase Ran/TC4 (4, 5). Ran alternates between a GTP-bound state and a GDP-bound state, a transition facilitated by a nuclear GTP-exchange factor (RCC1) and a cytoplasmic GTPase-activating protein (RanGAP1) (reviewed in Refs. 6 and 7). The differential binding of Ran-GTP and Ran-GDP to carrier proteins (e.g. importins, transportin, and NTF2) and NPC proteins provides a mechanism that allows Ran to regulate the transport of cargo across the nuclear pore complex. In this system, the subcellular localization of RCC1 and RanGAP1 are major determinants of directed nuclear transport.The localization of RanGAP1 to the nuclear pore complex is imparted by its specific association with Nup358/RanBP2 (1), a component of the cytoplasmic filaments emanating from the NPC (8, 9). This interaction requires the covalent modification of RanGAP1 with a ubiquitin-related protein designated SUMO-1 (1, 2). Recently, Ubc9, a structural homologue of the E2-type UBCs, was shown to coimmunoprecipitate with Nup358 in Xenopus egg extracts (3). Previous studies indicated that Ubc9 may be required for regulating the progression through the cell cycle in yeast (10,11). In addition, the human homologue of Ubc9 (HsUbc9) can functionally substitute for yeast Ubc9 (12,13). Although HsUbc9 has been proposed to mediate the ubiquitin-catalyzed degradation of mitotic cyclins (10 -12), no bona fide ubiquitin-conjugating activity has been demonstrated for HsUbc9.We now show that HsUbc9 differs significantly from the E2 family of UBCs in that it can form a thiolester intermediate with SUMO-1, rather than ubiquitin. This enzymatic activity is ATP-dependent and is not observed in mutants of HsUbc9, which contain a single amino acid substitution at the putative catalytic site. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. MATERIALS AND METHODS Preparation of Affinity Purified Antibodies-Rabbit antiserum raisedagainst Escherichia coli-derived recombinant HsUbc9 was affinity purified by passage over a CNBr-activated Sepharose B matrix coupled to E. coli-deri...

Histone sumoylation is associated with transcriptional repression

“…Indeed, the known SUMO ligase, PIAS (9-11) had no effect on H4 sumoylation in vivo or in vitro. These data are consistent with previous reports showing that, unlike ubiquitination, an E3 for sumoylation is not absolutely required, and some sumoylation substrates directly bind to E2 (UBC9) (32,33). We have also shown that the N-terminal tail of H4 is a target for linkage of multiple SUMO moieties (Fig.…”

Interaction and colocalization of PGP9.5 with JAB1 and p27Kip1

“…UBC9 is a ligase for an ubiquitin like protein, SUMO (Sampson et al, 2001). SUMO modi®cation is thought to be involved in the nuclear transport of the modi®ed proteins (Lee et al, 1998;Melchior, 2000). RanBPM is a newly identi®ed factor that interacts with Ran (Nishitani et al, 2001), a small GTP binding protein involved in nuclear tra c of many proteins (Izaurralde et al, 1997).…”

SUMO: of branched proteins and nuclear bodies

“…Unlike ubiquitylation, for which numerous ubiquitin conjugating enzymes (Ubc's) have been described, the SUMO pathway apparently uses only a single E2 enzyme, Ubc9 (Gong et al, 1997;Johnson and Blobel, 1999;Schwarz et al, 1998;Lee et al, 1998;Saitoh et al, 1998). Through a conserved Cys residue (Cys93), Ubc9 forms a thioester linkage with SUMO prior to conjugation with the target protein.…”