Posttranslational modification with small ubiquitin-related modifier (SUMO) proteins is now established as one of the key regulatory protein modifications in eukaryotic cells. Hundreds of proteins involved in processes such as chromatin organization, transcription, DNA repair, macromolecular assembly, protein homeostasis, trafficking, and signal transduction are subject to reversible sumoylation. Hence, it is not surprising that disease links are beginning to emerge and that interference with sumoylation is being considered for intervention. Here, we summarize basic mechanisms and highlight recent developments in the physiology of sumoylation.

Mammalian Bicaudal D2 is the missing molecular link between cytoplasmic motor proteins and the nucleus during nuclear positioning prior to the onset of mitosis.

Posttranslational modification with small ubiquitin-related modifier, SUMO, is a widespread mechanism for rapid and reversible changes in protein function. Considering the large number of known targets, the number of enzymes involved in modification seems surprisingly low: a single E1, a single E2, and a few distinct E3 ligases. Here we show that autosumoylation of the mammalian E2-conjugating enzyme Ubc9 at Lys14 regulates target discrimination. While not altering its activity toward HDAC4, E2-25K, PML, or TDG, sumoylation of Ubc9 impairs its activity on RanGAP1 and strongly activates sumoylation of the transcriptional regulator Sp100. Enhancement depends on a SUMO-interacting motif (SIM) in Sp100 that creates an additional interface with the SUMO conjugated to the E2, a mechanism distinct from Ubc9 approximately SUMO thioester recruitment. The crystal structure of sumoylated Ubc9 demonstrates how the newly created binding interface can provide a gain in affinity otherwise provided by E3 ligases.

In vertebrate cells, the nucleoporin Nup358/RanBP2 is a major component of the filaments that emanate from the nuclear pore complex into the cytoplasm. Nup358 forms a complex with SUMOylated RanGAP1, the GTPase activating protein for Ran. RanGAP1 plays a pivotal role in the establishment of a RanGTP gradient across the nuclear envelope and, hence, in the majority of nucleocytoplasmic transport pathways. Here, we investigate the roles of the Nup358-RanGAP1 complex and of soluble RanGAP1 in nuclear protein transport, combining in vivo and in vitro approaches. Depletion of Nup358 by RNA interference led to a clear reduction of importin ␣/␤-dependent nuclear import of various reporter proteins. In vitro, transport could be partially restored by the addition of importin ␤, RanBP1, and/or RanGAP1 to the transport reaction. In intact Nup358-depleted cells, overexpression of importin ␤ strongly stimulated nuclear import, demonstrating that the transport receptor is the most rate-limiting factor at reduced Nup358-concentrations. As an alternative approach, we used antibody-inhibition experiments. Antibodies against RanGAP1 inhibited the enzymatic activity of soluble and nuclear pore-associated RanGAP1, as well as nuclear import and export. Although export could be fully restored by soluble RanGAP, import was only partially rescued. Together, these data suggest a dual function of the Nup358-RanGAP1 complex as a coordinator of importin ␤ recycling and reformation of novel import complexes. INTRODUCTIONThe giant nucleoporin Nup358/RanBP2 (Wu et al., 1995;Yokoyama et al., 1995) is a major component of the filaments that emanate from the cytoplasmic ring of the nuclear pore complex (NPC) into the cytoplasm (Walther et al., 2002). As many other nucleoporins, Nup358 interacts via phenylalanine-glycine repeats (FG repeats) with karyopherins, transport receptors that mediate import and export across the NPC (for review see Tran and Wente, 2006). In recent nuclear transport models, a hydrophobic milieu that is established by FG repeats derived from several nucleoporins is suggested to allow selective translocation of karyopherins, with or without cargo molecules, across the NPC (for review see Weis, 2007). Individual nucleoporins do not play a major role in these models. Nevertheless, several nucleoporins have been shown to affect specific transport pathways. Nup153, for example, interacts with various import receptors, regulating late steps in nuclear import (Shah and Forbes, 1998). Very recently, specific FG nucleoporins were shown to be required for mRNA export (Terry and Wente, 2007). In this study, we investigate the role of Nup358 in nuclear protein transport in detail.Ran is a small GTP-binding protein that binds to karyopherins and plays an essential role in the majority of nucleocytoplasmic transport pathways. RanGTP is generated in the nucleus, resulting from the activity of the chromatinbound guanosine nucleotide exchange factor RCC1. In the cytoplasm, vertebrate RanGAP1 (RanGAP for short), together with the RanGTP-bind...