Posttranslational protein modification with small ubiquitinrelated modifier (SUMO) is an important regulatory mechanism implicated in many cellular processes, including several of biomedical relevance. We report that inhibition of the proteasome leads to accumulation of proteins that are simultaneously conjugated to both SUMO and ubiquitin in yeast and in human cells. A similar accumulation of such conjugates was detected in Saccharomyces cerevisiae ubc4 ubc5 cells as well as in mutants lacking two RING finger proteins, Ris1 and Hex3/Slx5-Slx8, that bind to SUMO as well as to the ubiquitin-conjugating enzyme Ubc4. In vitro, Hex3-Slx8 complexes promote Ubc4-dependent ubiquitylation. Together these data identify a previously unrecognized pathway that mediates the proteolytic down-regulation of sumoylated proteins. Formation of substrate-linked SUMO chains promotes targeting of SUMO-modified substrates for ubiquitin-mediated proteolysis. Genetic and biochemical evidence indicates that SUMO conjugation can ultimately lead to inactivation of sumoylated substrates by polysumoylation and/or ubiquitin-dependent degradation. Simultaneous inhibition of both mechanisms leads to severe phenotypic defects.Small ubiquitin-related modifier (SUMO), 5 which is structurally related to ubiquitin, is conjugated posttranslationally to a large number of substrates (1-5). The enzymes mediating SUMO conjugation are similar to those that catalyze the transfer of ubiquitin (3, 4). The functions of these two modifications, however, are distinct. Posttranslational modification of proteins with certain types of ubiquitin chains serves as a secondary degradation signal that targets such proteins for degradation by the 26 S proteasome (6). SUMO modification, in contrast, is not thought to result in proteolytic targeting (1-3, 7). Among the many functions of SUMO modification are regulation of transcription, nuclear transport, formation of subnuclear structures, cell cycle progression, and DNA repair (1-5, 7-9). Several substrates can be modified either by ubiquitin or SUMO (10). Modification of PCNA on a specific Lys residue by ubiquitylation mediates DNA repair (10, 11). Sumoylation of the same Lys, in contrast, mediates interaction with the Srs2 helicase, which results in inhibition of recombination during DNA replication (12, 13). In this example, sumoylation and ubiquitylation appear to direct proliferating cell nuclear antigen into distinct functions by promoting alternative interactions. Sumoylation of I B␣ on Lys 21 has been proposed to prevent its ubiquitylation and subsequent degradation (14). It has also been shown that SUMO-1 modification of a pathogenic fragment of Huntingtin enhances stability of this fragment, thereby increasing neurodegeneration, whereas ubiquitylation reduces fragment stability (15).Several recent studies suggested that, similar to ubiquitin, SUMO can form substrate-linked chains. In Saccharomyces cerevisiae, SUMO chain formation does not appear to serve an essential function (16). The reduced ability to remov...

The anaphase-promoting complex/cyclosome bound to CDC20 (APC/CCDC20) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) which inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which has been proposed to require CDC20 auto-ubiquitylation. Here we characterized APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C’s MCC binding site, is required for APC/CMCC-dependent CDC20 auto-ubiquitylation and degradation, and for timely anaphase initiation, but is dispensable for substrate ubiquitylation by APC/CCDC20 and APC/CCDH1. Our results support the view that MCC is continuously assembled and disassembled to enable rapid activation of APC/CCDC20 and that CDC20 auto-ubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators, and report the first generation of recombinant human APC/C.

The COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.