Abstract
The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C.
Highlights
Trypanosoma brucei, is an early divergent protozoan parasite that causes African sleeping sickness in human and nagana in livestock
We identified seven anaphase-promoting complex/cyclosome (APC/C) subunit homologues in T. brucei that include APC1, APC2, APC10/ DOC1, APC11, CDC16, CDC23 and CDC27 [23]
In order to clarify if some of the T. brucei APC/C subunits are capable of replacing the essential functions of their yeast counterparts, we performed yeast complementation assays to cover a total of 5 S. cerevisiae temperature sensitive mutants apc1-1, cdc27-1, cdc16-1, cdc23-1 and apc10-1 [29] with the corresponding T. brucei subunit homologues
Summary
Trypanosoma brucei, is an early divergent protozoan parasite that causes African sleeping sickness in human and nagana in livestock. Procyclic form cells can undergo cytokinesis in the absence of mitosis, whereas a mitotic arrest in bloodstream form cells inhibits cytokinesis with continued kinetoplast replication and segregation and nuclear DNA synthesis [2,5,6], which implicates fundamental differences in cell cycle controls between different life cycle forms of T. brucei and potential absence of the key cell cycle checkpoints Cell division in both forms of T. brucei proceeds longitudinally along the dorsal line from the anterior to the posterior end of the cell [7], which contrasts significantly from that in metazoa [8,9]. The cell cycle of T. brucei has become one of the most intriguing subjects for further investigation in recent years
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