The Rsp5 ubiquitin ligase contains a non-covalent binding site for ubiquitin within the amino-terminal lobe (N-lobe) of the HECT domain, and the X-ray crystal structure of the HECT-ubiquitin complex has been determined. Hydrophobic patch residues of ubiquitin (L8, I44, V70) were crucial for interaction with Rsp5, and amino-acid alterations at the Rsp5-binding interface resulted in defects in po...

The length of the ubiquitin chain on a substrate dictates various functional outcomes, yet little is known about its regulation in vivo. The yeast arrestin-related protein Rim8/Art9 is monoubiquitinated in vivo by the Rsp5 ubiquitin ligase. This also requires Vps23, a protein that displays an ubiquitin-E2 variant (UEV) domain. Here, we report that binding of the UEV domain to Rim8 interferes wi...

Figure 1. ‘Proteolytic’ and ‘non-proteolytic’ ubiquitin modifications in health and disease. Proteins modified by specific (e.g. lysine-48-linked) poly-ubiquitin chains (red) are normally targeted to degradation. ‘Non-proteolytic’ ubiquitin modifications are mono-ubiquitin and a non-canonical (lysine-63-linked) poly-ubiquitin chain. Malfunctions of the respective pathways can cause numerous hum...

The purification and biochemical characterization of protein substrates of the ubiquitin-dependent pathway of proteolysis is made difficult in part by the low steady state levels of ubiquitin-protein conjugates. We report here on the use of a polyhistidine-tagged ubiquitin molecule (HisUb) for the purification of ubiquitin-protein conjugates by metal chelate chromatography. When Escherichia col...

Previous studies in a cell-free proteolytic system from reticulocytes indicated that the conjugation of ubiquitin with proteins plays a role in protein breakdown. To examine some of the physiological functions of the ubiquitin conjugation system, and immunochemical method was developed for the isolation of ubiquitin-protein conjugates from intact cells. A specific antiserum was raised against u...

To construct a high information content assay for examination of the function of the cellular ubiquitin system, we added his-tagged ubiquitin, ATP, and an ATP-regenerating system to endogenous human cellular ubiquitin system enzymes, and labeled cellular proteins with hexa-histidine tagged ubiquitin in vitro. Labeling depended on ATP, the ATP recycling system, the proteasome inhibitor MG132, an...

BRCA1-associated protein 1 (BAP1) is a nuclear localizing UCH, having tumor suppressor activity and is widely involved in many crucial cellular processes. BAP1 has garnered attention for its links with cancer, however, the molecular mechanism in the regulation of cancer by BAP1 has not been established. Amongst the four UCHs, only BAP1 and UCHL5 are able to hydrolyze small and large ubiquitin a...

A role for ubiquitin in the pathogenesis of human diseases was first suggested some two decades ago, from studies that localized the protein to intracellular protein aggregates, which are a feature of the major human neurodegenerative disorders. Although several different mechanisms have been proposed to connect impairment of the UPS (ubiquitin-proteasome system) to the presence of these 'ubiqu...

Ubiquitin (Ub) modification plays an essential role in the regulation of various cellular processes. Ub performs a remarkable array of cellular tasks through the production of a large number of ubiquitinated proteins; such tasks require many Ubs. Ubs are expressed abundantly from several Ub encoding genes, though not in excess. Rather, Ub expression is tightly regulated through various control ...

Protein modification by ubiquitin and ubiquitin-like proteins is one of the most complex and intensely studied mechanisms of posttranslational protein regulation in eukaryotes. Conjugation of the 76-amino-acid protein ubiquitin is first and foremost a signal for targeting proteins to the proteasome for degradation, but evidence that ubiquitin also plays diverse roles in the regulation of numero...