APC16 is a conserved subunit of the anaphase-promoting complex/cyclosome

Abstract

SummaryError-free chromosome segregation depends on timely activation of the multi-subunit E3 ubiquitin ligase APC/C. Activation of the APC/C initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for destruction. The APC/C is the principle target of the mitotic checkpoint, which prevents segregation while chromosomes are unattached to spindle microtubules. We now report the identification and characterization of APC16, a conserved subunit of the APC/C. APC16 was found in association with tandem-affinity-purified mitotic checkpoint complex protein complexes. APC16 is a bona fide subunit of human APC/C: it is present in APC/C complexes throughout the cell cycle, the phenotype of APC16-depleted cells copies depletion of other APC/C subunits, and APC16 is important for APC/C activity towards mitotic substrates. APC16 sequence homologues can be identified in metazoans, but not fungi, by four conserved primary sequence stretches. We provide evidence that the C. elegans gene K10D2.4 and the D. rerio gene zgc:110659 are functional equivalents of human APC16. Our findings show that APC/C is composed of previously undescribed subunits, and raise the question of why metazoan APC/C is molecularly different from unicellular APC/C.

Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C)

“…Its requirement for APC/C assembly, and its location at the top of the TPR lobe [22], presumably close to Apc3/Cdc27, suggests an analogous role to the Cdc27-associated Apc9 subunit of S. cerevisiae APC/C [19]. Apc16 is required for the metaphase-toanaphase transition in human cells, and its loss stabilizes APC/C substrates in mitosis [23]. In the context of human cells, Apc16 was not required for the assembly of conserved APC/C subunits [23], raising the question of what role Apc16 plays to mediate APC/C-dependent substrate ubiquitination.…”

“…Interestingly Apc15 [33] and Apc16 [22,23] were only discovered as human APC/C subunits relatively recently. Our efforts to generate fully assembled recombinant human APC/C prior to the identification of Apc16 were unsuccessful.…”

“…Apc16 is required for the metaphase-toanaphase transition in human cells, and its loss stabilizes APC/C substrates in mitosis [23]. In the context of human cells, Apc16 was not required for the assembly of conserved APC/C subunits [23], raising the question of what role Apc16 plays to mediate APC/C-dependent substrate ubiquitination. Further emphasizing its importance, Apc16 is a conserved subunit of Caenorhabditis elegans and Danio rerio APC/C [23,50], although it does not share recognizable sequence similarities with yeast APC/C subunits.…”

“…The APC/C is highly conserved across eukaryotes, and 12 out of the 13 Saccharomyces cerevisiae APC/C subunits are conserved in Schizosaccharomyces pombe and humans ( Table 1). The latter two species lack Apc9 of S. cerevisiae APC/C, but instead incorporate the species-specific subunits Apc14 and Apc16 respectively [22,23]. Vertebrate APC/C also differs from yeast APC/C through an additional TPR subunit, Apc7, a paralogue of Apc3/Cdc27.…”

“…The ability to recapitulate the endogenous APC/C catalytic and regulatory activity using recombinant reconstituted APC/C provided strong evidence that the molecular composition of S. cerevisiae APC/C had been completely defined, a crucial prerequisite for understanding the complete system. Interestingly, despite the characterization of yeast and human APC/C subunits some 14 years ago [32], the human Apc15 [33] and Apc16 [22,23] subunits have only been identified relatively recently.…”