UC Berkeley

Posttranslational modification of proteins with ubiquitin, ubiquitylation, is essential in all eukaryotes. Ubiquitylation is accomplished by an enzymatic cascade: E1 activates ubiquitin and transfers it to E2, which works with E3 to transfer ubiquitin to substrate residues, usually lysines. Once attachment of the first ubiquitins to substrate, chain initiation, has occurred, E2 and E3 modify one of the seven lysine residues in ubiquitin itself, resulting in formation of polymeric ubiquitin chains, chain elongation. Ubiquitylation is involved in nearly every cellular process, and is central to cell cycle control. Ubiquitin-dependent protein degradation is especially important during mitosis, as cells must turn over many proteins within a short time-window. The anaphase-promoting complex (APC/C) is an essential E3 that targets cell cycle regulators for degradation during mitosis. The APC/C must degrade many substrates within a short period of time, and likely acts under saturation. Furthermore, APC/C substrates are required at different times during mitosis. Therefore, the APC/C cannot degrade all substrate at once, but rather must order substrate degradation so that substrate proteins can accomplish their functions. The balance between efficiency and regulated degradation is the APC/C's major challenge as it orchestrates mitotic exit.

In this dissertation I address how human APC/C targets its many substrates for degradation. First, I report our finding that APC/C functions by decorating its substrates with Lys11-linked ubiquitin chains. Lys11-linked chains target substrates to the 26S proteasome and are essential for cell division in human cells, Drosophila cells, and Xenopus tropicalis embryos. The human APC/C uses a dedicated E2, Ube2C/UbcH10, to perform chain initiation while a second E2, Ube2S, elongates these first modifications using exclusively Lys11 on ubiquitin. Ube2C and Ube2S likely function simultaneously, thus allowing APC/C to overcome its massive substrate workload. Efficient chain initiation by Ube2C is made possible by conserved initiation motifs in substrates. Initiation motifs do not regulate substrate binding to APC/C, but instead control the efficiency with which Ube2C and APC/C attach the first ubiquitin moieties to substrates. I will show that chain initiation by Ube2C is the rate-limiting, regulated step during Lys11-linked chain formation by the APC/C. Once Ube2C has initiated chains on APC/C substrates, Ube2S takes over, rapidly elongating Lys11-linked ubiquitin chains that target substrates to the 26S proteasome. Finally, I address how use of two E2 enzymes performing different functions allows APC/C to overcome its major challenge: rapid yet regulated turnover of essential cell cycle regulators. The separation of ubiquitin chain formation by the APC/C into initiation and elongation steps, with initiation being rate-limiting and tightly regulated, draws parallels between ubiquitylation and other processive reactions such as transcription and translation.