THE TAIL OF A UBIQUITIN-CONJUGATING ENZYME REDIRECTS MULTI-UBIQUITIN CHAIN SYNTHESIS FROM THE LYSINE 48-LINKED CONFIGURATION TO A NOVEL NONLYSINE-LINKED FORM

The UBC1 ubiquitin-conjugating enzyme from Saccharomyces cerevisiae ha s an overlapping function with the UBC4 and UBC5 enzymes in the yeast stress response and an important role in the G(0) to G(1) transition t hat accompanies spore germination (Seufert, W., McGrath, J. P., and Je ntsch, S. (1990) EMBO J. 9, 4573-4541). In the present work we report that the UBC1 enzyme assembles onto itself a multi-ubiquitin chain in vitro whose linkage configuration is dependent on the unconserved carb oxyl-terminal extension or tail that is appended to its catalytic doma in. Using chemical cleavage and site-specific mutagenesis, we have map ped the location of the chain to lysine 93 which lies near the active site within the catalytic domain The ubiquitin molecule that anchors t he chain is transferred to this lysine from the active site of the sam e UBC1 molecule. When the tail of UBC1 is deleted, the catalytic-domai n synthesizes a chain that consists of ubiquitin molecules uniformly l inked to one another via lysine 48. In the presence of the tail, howev er, a chain is assembled that is composed of linkages that are stable to alkali but which do not utilize lysines. Furthermore, when the amin o terminus of ubiquitin is blocked by an appended peptide tag, chain a ssembly reverts from this alternative configuration to the canonical l ysine 48 variety. Taken together, these results suggest that the alter native chain is composed of linkages in which one ubiquitin molecule f orms a peptide bond with the alpha-amino terminus of another, thereby supporting the emerging view that Ub can be attached to itself or othe r proteins in a variety of ways.