In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.

SUMMARYUbiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING of RNF4 in complex with E2 (UbcH5a) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The C-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilise the consequent tetrahedral transition state intermediate.

Conjugation of the small ubiquitin-like modifier SUMO-1/SMT3C/Sentrin-1 to proteins in vitro is dependent on a heterodimeric E1 (SAE1/SAE2) and an E2 (Ubc9). Although SUMO-2/SMT3A/Sentrin-3 and SUMO-3/SMT3B/Sentrin-2 share 50% sequence identity with SUMO-1, they are functionally distinct. Inspection of the SUMO-2 and SUMO-3 sequences indicates that they both contain the sequence KXE, which represents the consensus SUMO modification site. As a consequence SAE1/ SAE2 and Ubc9 catalyze the formation of polymeric chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detected in vivo. The ability to form polymeric chains is not shared by SUMO-1, and although all SUMO species use the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences.The small ubiquitin-like modifier SUMO-1 1 (also known as SMT3C, Sentrin, GMP1, UBL1, and PIC1) is a member of the ubiquitin-like protein family (1). SUMO-1 is known to be covalently conjugated to a variety of cellular substrates via a three-step enzymatic pathway analogous to that of ubiquitin conjugation. The E1-like enzymes for both SUMO-1 and the yeast homologue Smt3p exist as heterodimers known as SAE1/ SAE2 and Uba2p/Aos1p, respectively (2-5). In the first step the SAE1/SAE2 heterodimer utilizes ATP to adenylate the C-terminal glycine of SUMO-1. Formation of a thioester bond between the C-terminal glycine of SUMO-1 and a cysteine residue in SAE2 is accompanied by the release of AMP. The second step is a transesterification reaction, which transfers SUMO-1 from the E1 to a cysteine residue within the SUMO-specific E2-conjugating enzyme (Cys 93 in Ubc9). In the third step, Ubc9 catalyzes the formation of an isopeptide bond between the C terminus of SUMO-1 and the ⑀-amino group of lysine in the target protein. In contrast to the ubiquitin conjugation pathway no activity equivalent to an E3 ligase is required for SUMO-1 conjugation in vitro (2, 4), suggesting that the specificity for target proteins is conferred by Ubc9 itself or the Ubc9⅐SUMO-1 thioester complex. This is supported by the observations that almost all SUMO-1-conjugated proteins bind Ubc9 in two-hybrid assays, and the acceptor lysine residues on target proteins appear to exist within the consensus motif KXE (where represents a large hydrophobic amino acid, and X represents any amino acid) (6 -8). Furthermore, SUMO-1 is thought not to form SUMO-1-SUMO-1 polymers, which are characteristic of ubiquitination.Unlike the majority of ubiquitinated proteins, acceptors of SUMO-1 modifications are not targeted for degradation. In fact, in the case of the transcriptional inhibitor IB␣ the target lysine for SUMO-1 modification is the same as that of ubiquitin conjugation, thus blocking ubiquitination at that residue and stabilizing the protein (8). Transcriptional activity of specific proteins appears to be affected by SUMO-1 modification. For example, conjugation at a single site in the C terminus of p53 activates its transcriptional response (9, 10)...

Covalent conjugation of the small ubiquitin-like modifier (SUMO) proteins to target proteins regulates many important eukaryotic cellular mechanisms. Although the molecular consequences of the conjugation of SUMO proteins are relatively well understood, little is known about the cellular signals that regulate the modification of their substrates. Here, we show that SUMO-2 and SUMO-3 are required for cells to survive heat shock. Through quantitative labeling techniques, stringent purification of SUMOylated proteins, advanced mass spectrometric technology, and novel techniques of data analysis, we quantified heat shock-induced changes in the SUMOylation state of 766 putative substrates. In response to heat shock, SUMO was polymerized into polySUMO chains and redistributed among a wide range of proteins involved in cell cycle regulation; apoptosis; the trafficking, folding, and degradation of proteins; transcription; translation; and DNA replication, recombination, and repair. This comprehensive proteomic analysis of the substrates of a ubiquitin-like modifier (Ubl) identifies a pervasive role for SUMO proteins in the biologic response to hyperthermic stress.

The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.

Posttranslational modification with small ubiquitin-like modifiers (SUMOs) alters the function of proteins involved in diverse cellular processes. SUMO-specific enzymes conjugate SUMOs to lysine residues in target proteins. Although proteomic studies have identified hundreds of sumoylated substrates, methods to identify the modified lysines on a proteomic scale are lacking. We developed a method that enabled proteome-wide identification of sumoylated lysines that involves the expression of polyhistidine (6His)-tagged SUMO2 with Thr(90) mutated to Lys. Endoproteinase cleavage with Lys-C of 6His-SUMO2(T90K)-modified proteins from human cell lysates produced a diGly remnant on SUMO2(T90K)-conjugated lysines, enabling immunoprecipitation of SUMO2(T90K)-modified peptides and producing a unique mass-to-charge signature. Mass spectrometry analysis of SUMO-enriched peptides revealed more than 1000 sumoylated lysines in 539 proteins, including many functionally related proteins involved in cell cycle, transcription, and DNA repair. Not only can this strategy be used to study the dynamics of sumoylation and other potentially similar posttranslational modifications, but also, these data provide an unprecedented resource for future research on the role of sumoylation in cellular physiology and disease.

The small ubiquitin-like modifiers (SUMOs) alter the functions of diverse cellular proteins by covalent posttranslational modification and thus influence many cellular functions, including gene transcription, cell cycle, and DNA repair. Although conjugation by ubiquitin and SUMO-2/3 are largely functionally and mechanistically independent from one another, both appear to increase under conditions of proteasome inhibition. To better understand the relationship between SUMO and protein degradation by the proteasome, we performed a quantitative proteomic analysis of SUMO-2 substrates after short- and long-term inhibition of the proteasome with MG132. Comparisons with changes to the SUMO-2 conjugate subproteome in response to heat stress revealed qualitative and quantitative parallels between both conditions; however, in contrast to heat stress, the MG132-triggered increase in SUMO-2 conjugation depended strictly on protein synthesis, implying that the accumulation of newly synthesized, misfolded proteins destined for degradation by the proteasome triggered the SUMO conjugation response. Furthermore, proteasomal inhibition resulted in the accumulation of conjugated forms of all SUMO paralogs in insoluble protein inclusions and in the accumulation on SUMO-2 substrates of lysine-63-linked polyubiquitin chains, which are not thought to serve as signals for proteasome-mediated degradation. Together, these findings suggest multiple, proteasome-independent roles for SUMOs in the cellular response to the accumulation of misfolded proteins.

The small ubiquitin-like modifier (SUMO) can undergo selfmodification to form polymeric chains that have been implicated in cellular processes such as meiosis, genome maintenance and stress response. Investigations into the biological role of polymeric chains have been hampered by the absence of a protocol for the purification of proteins linked to SUMO chains. In this paper, we describe a rapid affinity purification procedure for the isolation of endogenous polySUMO-modified species that generates highly purified material suitable for individual protein studies and proteomic analysis. We use this approach to identify more than 300 putative polySUMO conjugates from cultured eukaryotic cells.