Investigation of mixed ubiquitin chains and chain conformations

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Ubiquitination is an essential posttranslational modification regulating widespread intracellular processes in eukaryotic cells. Covalent modification of target molecules with ubiquitin is not a simple on-off information like phosphorylation, but can occur in several distinct modes. A key question in the field is to understand how the ubiquitin code is generated and how it is decoded by downstream acting factors. Versatility of the ubiquitin code is achieved by different types of modifications of target proteins: They can either be modified with single ubiquitin molecules at specific sites (mono-ubiquitination), with multiple ubiquitin molecules at several sites (multi mono-ubiquitination) or with various forms of polyubiquitin chains. Chains of different linkage type are associated with different functions. For example, ubiquitin chains of at least four K48-linked ubiquitin moieties mark cytosolic substrates for proteasomal degradation. K11-linked chains have been shown to play important roles in Endoplasmic Reticulum Associated Protein Degradation (ERAD) and cell cycle control, whereas K63-linked chains can mediate widespread non-proteasomal functions such as DNA repair, transcriptional activation, endocytosis or protein trafficking. So far ubiquitin research has focused primarily on the study of homogeneously linked polyubiquitin chains. However there is growing evidence that chains containing more than one linkage type exist in vivo either in form of mixed-linkage-chains or branched chains. For instance K63/M1, K11/K48 and K63/K48 branches have already been detected in cells. By studying ubiquitination processes in the ERAD system we have discovered that the E2 enzyme Ubc1 is decorating K63-linked substrates with K48-linkages thereby introducing K48/K63 branches. Using a combination of yeast genetics and biochemical as well as biophysical methods we want to characterize this reaction further, detect K48/K63 linkages in vivo, identify UBDs specifically binding branched chains, characterize these interactions functionally and structurally and investigate the conformations that different ubiquitin chains can adopt.