Analysis of cellular SUMO and SUMO-ubiquitin hybrid conjugates.

Posttranslational modification of proteins with the small ubiquitin-related modifier (SUMO) has been implicated in many important physiological functions, including the regulation of transcription and DNA repair. In most cases, only a small fraction of the total cellular amounts of a given protein is sumoylated at a certain point in time. Sensitive detection of sumoylated forms of proteins by western blotting is, therefore, an important step in the identification and/or characterization of a protein control by sumoylation.

Arsenic trioxide stimulates SUMO-2/3 modification leading to RNF4-dependent proteolytic targeting of PML.

We have recently reported that poly-SUMO-2/3 conjugates are subject to a ubiquitin-dependent proteolytic control in human cells. Here we show that arsenic trioxide (ATO) increases SUMO-2/3 modification of promyelocytic leukemia (PML) leading to its subsequent ubiquitylation in vivo. The SUMO-binding ubiquitin ligase RNF4 mediates this modification and causes disruption of PML nuclear bodies upon treatment with ATO.

Ubiquitin-dependent proteolytic control of SUMO conjugates.

Posttranslational protein modification with small ubiquitin-related 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.