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 has an overlapping function with the UBC4 and UBC5 enzymes in the yeast stress response and an important role in the G0 to G1 transition that accompanies spore germination (Seufert, W., McGrath, J. P., and Jentsch, 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 carboxyl-terminal extension or tail that is appended to its catalytic domain. Using chemical cleavage and site-specific mutagenesis, we have mapped the location of the chain to lysine 93 which lies near the active site within the catalytic domain. The ubiquitin molecule that anchors the chain is transferred to this lysine from the active site of the same UBC1 molecule. When the tail of UBC1 is deleted, the catalytic domain synthesizes a chain that consists of ubiquitin molecules uniformly linked to one another via lysine 48. In the presence of the tail, however, a chain is assembled that is composed of linkages that are stable to alkali but which do not utilize lysines. Furthermore, when the amino terminus of ubiquitin is blocked by an appended peptide tag, chain assembly reverts from this alternative configuration to the canonical lysine 48 variety. Taken together, these results suggest that the alternative chain is composed of linkages in which one ubiquitin molecule forms a peptide bond with the alpha-amino terminus of another, thereby supporting the emerging view that Ub can be attached to itself or other proteins in a variety of ways.
Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada. Mike_Ellison@darwin.biochem.ualberta.ca
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D001665:Binding Sites; D008239:Lysine / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014452:Ubiquitins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D014452:Ubiquitins; D008239:Lysine