© 2000 Macmillan Magazines Ltd review NATURE CELL BIOLOGY | VOL 2 | AUGUST 2000 | www.nature.com/ncb E153 Evolution and function of ubiquitin-like protein-conjugation systems Mark Hochstrasser Department of Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Avenue, New Haven, Connecticut 06520, USA e-mail: mark.hochstrasser@yale.edu Ubiquitin functions by covalently modifying other proteins. In the past few years, a surprising number of other proteins have been identified that, despite often being only slightly similar to ubiquitin, can also be attached to proteins. Newly discovered parallels between the activation of ubiquitin and the biosynthesis of certain enzyme cofactors now hint at the possible evolutionary origins of the ubiquitin system. ellular proteins are frequently adorned with chemical modifica- tions that alter their physical —and, as a result, their physiolog- ical — properties. Among the most spectacular of these molecular appendages are grafts of other proteins or even polymeric chains of other proteins. The prototypical example of an intracellular polypeptide that covalently modifies another protein is ubiquitin. Ubiquitin is one of the most phylogenetically well-conserved of all proteins in eukaryotes, but is curiously absent from any archaeal or eubacterial species for which the full genome sequence is known. Most frequently, the ubiquitin tag is used to mark particular proteins for proteolytic elimination, but it can also have nonproteolytic func- tions. In the past several years, we have witnessed the discovery of a surprising number of other ubiquitin-like modifiers (Ubls), which all resemble ubiquitin in their mechanisms of substrate conjugation and which are turning out to have a wide range of functions 1–3 (Fig. 1). The prevalence of Ubls indicates that attachment of proteins to other proteins is a far more widely employed regulatory strategem than had initially been suspected. What might the advantages of Ubl addition be over other types of protein alteration such as phos- phorylation or acetylation? Being proteins, Ubls sport larger and more chemically varied surfaces than other types of modification, thus lending themselves to the modulation of protein conformation and interactions — even simultaneous interactions with different macromolecules. For example, the polyubiquitin chains that are assembled on many proteolytic substrates provide a matrix of bind- ing sites for specific and high-affinity adhesion to the proteasome 4 . This binding is largely independent of the substrate itself, allowing tethering of the conjugate while the substrate moiety is unfolded and degraded. De-ubiquitinating enzymes can recycle the ubiquitin from its doomed partner even as the latter is being fragmented. This dynamic character is common to many other Ubl–protein modifi- cations (Fig. 1). There also exists a class of ubiquitin-related pro- teins in which a ubiquitin domain (UbD) is built into a larger polypeptide and is not excised or attached to other proteins. These UbDs may impart properties to a protein that are analogous to those conferred by a transferable Ubl, even though the UbD is locked onto a specific target. Ubiquitin and its kin are linked to other proteins by an amide bond between the carboxy-terminal carboxylate of the modifier and one or more lysine side chains of the target protein 1 (Figs 1, 2a). In all cases, the C-terminal carboxyl group of the Ubl, which usually ends with a pair of glycine residues, is first activated by adenylation. A thiol group of the activating enzyme (E1) then attacks the car- boxyl–AMP of the Ubl to yield an E1–Ubl thioester. The E1 trans- fers its activated Ubl to the cysteine side chain of a member of a second family of proteins (the E2s), from which the Ubl is passed to a substrate lysine. A third enzyme (E3) is often required for this final transfer. E3 functions as an adaptor and/or allosteric activator for the E2, or is itself first linked to the Ubl by a thioester bond before finally yielding it to the substrate. Ubl function in the RUB1 and SUMO pathways Several new Ubl-modification systems have been discovered in the past few years. Of these, the SUMO (small-ubiquitin-related modi- fier) and RUB1 (related-to-ubiquitin 1) pathways have received the most intense scrutiny. The first target of SUMO to be identified was the nucleocytoplasmic-transport protein RanGAP1, the localiza- tion of which to nuclear-pore complexes requires ligation of SUMO to a specific lysine residue in the protein 5 . Genetic results strongly support a requirement for SUMO conjugation in the nuclear import of at least some proteins. In Drosophila, most of the embry- onic morphogen Bicoid is imported into the nucleus, where it func- tions as a DNA-binding transcriptional regulator. This import depends on a functional SUMO-conjugation system 6 . Potentially, sumoylation of RanGAP1 or of another component of the transport machinery is required for Bicoid import, or it may be that a SUMO- conjugated form of Bicoid is imported more efficiently (or exported less efficiently). Whether Bicoid is directly modified by SUMO is not yet known. In the past few years, several more SUMO substrates have been found and quite a few more can be anticipated. The consequences of these modifications are just beginning to be unravelled. One fre- quently voiced proposition is that addition of SUMO alters the localization, conformation or protein interactions of its targets. This seems to be true not only for sumoylated RanGAP1 but also for a group of proteins that localize to discrete intranuclear sites called C Figure 1 Reversible ligation of ubiquitin and Ubls to other proteins. A lysine side chain is generally the site of formation of an amide (isopeptide) bond with the C terminus of the activated Ubl. Activation and conjugation of Ubls requires a series of enzymes that act in sequence — E1, E2, and sometimes an E3 (see Fig. 2a). The Ubl–substrate conjugate has biological properties that are distinct from those of the unconjugated substrate, and the amount of protein in each state is determined by the relative rates of Ubl conjugation and deconjugation. DUB, de-ubiquitinating enzyme; ULP, Ubl-specific protease. NH ε NH 3 + Protein CO 2 – CO 2 – DUB ULP E1 E2 (E3) Ubl C=O Ubl–protein conjugate