RESUMO
Transient interactions between the anaphase-promoting complex/cyclosome (APC/C) and its activator subunit Cdc20 or Cdh1 generate oscillations in ubiquitylation activity necessary to maintain the order of cell cycle events. Activator binds the APC/C with high affinity and exhibits negligible dissociation kinetics in vitro, and it is not clear how the rapid turnover of APC/C-activator complexes is achieved in vivo. Here, we describe a mechanism that controls APC/C-activator interactions based on the availability of substrates. We find that APC/C-activator dissociation is stimulated by abundant cellular polyanions such as nucleic acids and polyphosphate. Polyanions also interfere with substrate ubiquitylation. However, engagement with high-affinity substrate blocks the inhibitory effects of polyanions on activator binding and APC/C activity. We propose that this mechanism amplifies the effects of substrate affinity on APC/C function, stimulating processive ubiquitylation of high-affinity substrates and suppressing ubiquitylation of low-affinity substrates.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/imunologia , Proteínas Cdc20/metabolismo , Proteínas Cdh1/metabolismo , Proteínas Fúngicas/metabolismo , Polímeros/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/isolamento & purificação , Proteínas Cdc20/isolamento & purificação , Proteínas Cdh1/isolamento & purificação , Ciclo Celular , Proteínas Fúngicas/isolamento & purificação , Ácidos Nucleicos/metabolismo , Polieletrólitos , Polifosfatos/metabolismo , Saccharomycetales/metabolismo , Especificidade por Substrato , UbiquitinaçãoRESUMO
The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated cells. In proliferating cells, APC/C controls cell cycle progression by targeting inhibitors of chromosome segregation and mitotic exit for degradation by the 26S proteasome. To understand how APC/C recruits and ubiquitylates its substrate proteins and how these processes are controlled, it is essential to analyze APC/C activity in vitro. In the past, such experiments have been limited by the fact that large quantities of purified APC/C were difficult to obtain and that mutated versions of the APC/C could not be easily generated. In this chapter we review recent advances in generating and purifying recombinant forms of the human APC/C and its co-activators, using methods that are scalable and compatible with mutagenesis. We also describe a method that allows the quantitative analysis of APC/C activity using fluorescently labeled substrate proteins.
