Mechanisms of ubiquitin transfer by the anaphase-promoting complex

The anaphase-promoting complex (APC) is a ubiquitin-protein ligase required for the completion of mitosis in all eukaryotes. Recent mechanistic studies reveal how this remarkable enzyme combines specificity in substrate binding with flexibility in ubiquitin transfer, thereby allowing the modification of multiple lysines on the substrate as well as specific lysines on ubiquitin itself

E2s Act Sequentially in the Assembly of Ubiquitin Chains by the Anaphase-Promoting Complex

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

Sequential E2s Drive Polyubiquitin Chain Assembly on APC Targets

SummaryThe anaphase-promoting complex (APC), or cyclosome, is an E3 ubiquitin-protein ligase that collaborates with E2 ubiquitin-conjugating enzymes to assemble polyubiquitin chains on proteins important for cell-cycle progression. It remains unclear how the APC—or many other E3s—promotes the multiple distinct reactions necessary for chain assembly. We addressed this problem by analyzing APC interactions with different E2s. We screened all budding yeast E2s as APC coenzymes in vitro and found that two, Ubc4 and Ubc1, are the key E2 partners for the APC. These proteins display strikingly different but complementary enzymatic behaviors: Ubc4 supports the rapid monoubiquitination of multiple lysines on APC targets, while Ubc1 catalyzes K48-linked polyubiquitin chain assembly on preattached ubiquitins. Mitotic APC function is lost in yeast strains lacking both Ubc1 and Ubc4. E2-25K, a human homolog of Ubc1, also promotes APC-dependent chain extension on preattached ubiquitins. We propose that sequential E2 proteins catalyze K48-linked polyubiquitination and thus proteasomal destruction of APC targets

Sgt1p and Skp1p Modulate the Assembly and Turnover of CBF3 Complexes Required for Proper Kinetochore Function

Kinetochores are composed of a large number of protein complexes that must be properly assembled on DNA to attach chromosomes to the mitotic spindle and to coordinate their segregation with the advance of the cell cycle. CBF3 is an inner kinetochore complex in the budding yeast Saccharomyces cerevisiae that nucleates the recruitment of all other kinetochore proteins to centromeric DNA. Skp1p and Sgt1p act through the core CBF3 subunit, Ctf13p, and are required for CBF3 to associate with centromeric DNA. To investigate the contribution of Skp1p and Sgt1p to CBF3 function, we have used a combination of in vitro binding assays and a unique protocol for synchronizing the assembly of kinetochores in cells. We have found that the interaction between Skp1p and Sgt1p is critical for the assembly of CBF3 complexes. CBF3 assembly is not restricted during the cell cycle and occurs in discrete steps; Skp1p and Sgt1p contribute to a final, rate-limiting step in assembly, the binding of the core CBF3 subunit Ctf13p to Ndc10p. The assembly of CBF3 is opposed by its turnover and disruption of this balance compromises kinetochore function without affecting kinetochore formation on centromeric DNA