BOS-hDDIT3 and pcDNA3NH2-hFUS were generated by cloning the corresponding cDNAs into the expression plasmids

y is inhibited. It is probable that the polyubiquitinated CycB2/cyc6 is a product of the APC/C action. Discussion 19111597 In the present study, we indicated that among the five APC/C subunit homologues in T. brucei; APC1, CDC16, CDC23, CDC27 and APC10, none was capable of complementing the function of their counterparts in yeast. Although APC/C has a conserved presence and function among all the eukaryotes examined thus far, the sequences of individual APC/C subunit proteins do not appear to be highly conserved. Drosophila APC11, C. elegans CDC26 and human APC13 have been, however, tested in the yeast complementation assays and found capable of substituting the corresponding subunits in yeast. It makes thus the negative outcome from testing all 5 T. brucei subunits a little difficult to explain from a simple view on protein sequence discrepancies. An alternative explanation could be by postulating a distinctive mechanism of inter-subunit interactions in constituting the APC/C in T. brucei, i.e., T. brucei APC/C subunits may be incapable of incorporating into yeast APC/C. The assembly and three-dimensional structure of APC/C remain poorly understood for the time being. Threedimensional electron microscopic structural analysis of yeast APC/C located APC1 in an L-shaped rod that links APC2 to CDC23, whereas CDC23 is connected to APC5 with APC4 interconnecting APC1 and APC5. APC1, the largest APC/C subunit, consists of 11 highly repetitive 22431203 3540 amino acid proteasome-cyclosome sequences at the C-terminus. This PC motif is shared with the RPN1 and RPN2 subunits of the proteasome 19S regulatory particle, and is assumed to be the main binding sites for other subunits in forming APC/C. APC/C purified from the yeast mutant apc1D was found lacking association among the majority of other subunits. APC1 is The APC/C of Trypanosoma brucei thus classified as the major scaffold protein in yeast APC/C. These 11 PC AG-1478 chemical information repeats are, however, absent from T. brucei APC1, which could provide a supporting evidence that T. brucei APC/C may have a mechanism of assembly highly distinctive from that in yeast. This distinction may explain why the APC/C subunits from T. brucei are not complementary to the corresponding subunits missing from the yeast. The outcome from our TAP and LC-MS/MS analysis of APC1-PTP protein complex indicates that T. brucei APC/C is made of 10 core subunit proteins. The composition and level of APC/C remain apparently unchanged throughout the entire cell cycle of procyclic-form T. brucei. An even more intriguing finding was that neither CDC20 nor MCC complex proteins were found associated with APC/C during any phase of the cell cycle. This is in contrast to that observed among the other eukaryotes. An intact MCC complex with CDC20 protein had been co-purified with APC/C subunits using a similar TAP procedure in human cells. Also, CDC20 was detected in the mitotic-enriched APC/CTAP sample from the budding yeast and fission yeast. In budding yeast, the MCC components MAD1, MAD2 and MAD3 were co-immunoprecipitated with epitope-tagged CDC20 throughout different stages of the cell cycle. A common factor enabling all the complex formations mentioned above is CDC20. The fact that the T. brucei CDC20 homologue is not associated with APC/C at all during all phases of the cell cycle shows that it is not performing the function of mediating a binding of APC/C to MCC or activating APC/C to poly-ubiquitinate mitotic cyclin CycB2/cyc6. Our previous finding that an

Leave a Reply