UCSF

Ubiquitination is used as a signaling mechanism by many processes in the cell. The ubiquitin signal can encode changes in intracellular trafficking, binding partners or protein turnover. Ubiquitin is a 76 amino acid protein that is covalently attached to other proteins via its C-terminus. The ubiquitination of proteins involves a cascade of three enzymes whose functions are to activate ubiquitin and attach it to lysine side chains of proteins. The last member of the cascade is an E3, or ubiquitin-protein ligase, that brings together substrates and ubiquitin-charged E2s. E3s allow for the transfer of ubiquitin from a catalytic cysteine on the E2 to a lysine side chain on the substrate. If lysines on ubiquitin itself are used during ubiquitination then polyubiquitin chains are built on the substrates. The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that assembles polyubiquitin chains on substrates important for cell cycle progression. In budding yeast, the APC collaborates with two E2 ubiquitin-conjugating enzymes, Ubc4 and Ubc1. We have demonstrated that Ubc4 and Ubc1 have very different enzymatic behaviors: APC reactions with Ubc4 result in rapid monoubiquitination of multiple nonspecific lysines on the substrate, whereas APC reactions with Ubc1 result in the assembly of polyubiquitin chains that are linked specifically through lysine 48 (K48) of ubiquitin. We have also found two residues, threonine 84 and glutamine 122, on Ubc1 that are required for K48-linked polyubiquitination. Both of these residues are on flexible loops near the catalytic cysteine, in a position where they could influence catalysis. Threonine 84 is involved in catalyzing K48-dependent ubiquitination, whereas glutamine 122 is involved in reducing the ubiquitination of nonspecific lysines on the substrate. We propose that Ubc1 is able to catalyze the assembly of K48-specific chains at the expense of substrate lysine ubiquitination.