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Structurally Related TPR Subunits Contribute Differently to the Function of the Anaphase-Promoting Complex in Drosophila Melanogaster

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Structurally Related TPR Subunits Contribute Differently to the Function of the Anaphase-Promoting Complex in Drosophila Melanogaster 3238 Research Article Structurally related TPR subunits contribute differently to the function of the anaphase-promoting complex in Drosophila melanogaster Margit Pál, Olga Nagy, Dalma Ménesi, Andor Udvardy and Péter Deák* Institute of Biochemistry, Biological Research Center, H-6726 Szeged, Hungary *Author for correspondence (e-mail: [email protected]) Accepted 10 July 2007 Journal of Cell Science 120, 3238-3248 Published by The Company of Biologists 2007 doi:10.1242/jcs.004762 Summary The anaphase-promoting complex/cyclosome or APC/C is causes different pupal lethality. Cytological examination a key regulator of chromosome segregation and mitotic exit showed that these animals had an elevated level of in eukaryotes. It contains at least 11 subunits, most of apoptosis, high mitotic index and delayed or blocked which are evolutionarily conserved. The most abundant mitosis in a prometaphase-metaphase-like state with constituents of the vertebrate APC/C are the four overcondensed chromosomes. The arrested neuroblasts structurally related tetratrico-peptide repeat (TPR) contained elevated levels of cyclin B but, surprisingly, subunits, the functions of which are not yet precisely cyclin A appeared to be degraded normally. Contrary to understood. Orthologues of three of the TPR subunits have the situation for the Apc6/Cdc16 and Apc8/Cdc23 genes, the been identified in Drosophila. We have shown previously apparent loss of Apc7 function does not lead to the above that one of the TPR subunits of the Drosophila APC/C, abnormalities. Instead, the Apc7 knocked down animals Apc3 (also known as Cdc27 or Mákos), is essential for and null mutants are viable and fertile, although they development, and perturbation of its function results in display mild chromosome segregation defects and mitotic cyclin accumulation and metaphase-like arrest. In anaphase delay. Nevertheless, the Apc7 subunit shows this study we demonstrate that the Drosophila APC/C synergistic genetic interaction with Apc8/Cdc23 that, associates with a new TPR protein, a genuine orthologue of together with the phenotypic data, assumes a limited the vertebrate Apc7 subunit that is not found in yeasts. In functional role for Apc7. Taken together, these data suggest addition to this, transgenic flies knocked down for three of that the structurally related TPR subunits contribute the TPR genes Apc6 (Cdc16), Apc7 and Apc8 (Cdc23), by differently to the function of the anaphase-promoting Journal of Cell Science RNA interference were established to investigate their complex. function. Whole-body expression of subunit-specific dsRNA efficiently silences these genes resulting in only residual mRNA concentrations. Apc6/Cdc16 and Key words: APC/C, TPR subunits, Apc6 (Cdc16), Apc7, Apc8 Apc8/Cdc23 silencing induces developmental delay and (Cdc23), Transgenic RNAi Introduction identified in higher eukaryotes as well, indicating that the The ubiquitin-dependent protein degradation system controls structure of APC/C is evolutionarily conserved. Only the Apc9 mitotic progression by sequential degradation of different subunit appears to be specific to unicellular yeasts, whereas the regulatory proteins. In this process a pathway of ubiquitin Apc7 subunit has so far been identified only in vertebrates activating (E1), ubiquitin conjugating (E2) and ubiquitin ligase (Harper et al., 2002). (E3) enzymes catalyse the conjugation of polyubiquitin chains Despite the essential role of APC/C in regulating mitosis, little onto substrate proteins, leading to their recognition and is known about the function of most of its individual subunits. degradation by the 26S proteasome. The key component of this The presence of structurally related proteins to the cullin proteolytic system is a multi-subunit ubiquitin ligase, the homologue Apc2, the RING finger Apc11 and the DOC1 anaphase-promoting complex (APC/C) that provides a domain containing Apc10 subunits in other E3 ubiquitin ligases platform and specificity for the ubiquitination reactions. The suggests direct roles of these subunits in the ubiquitination APC/C is active during mitosis and G1 phases of the cell cycle reaction. Indeed, it was shown that the Apc2 and Apc11 subunits in which, by mediating securin and mitotic cyclin degradation, interact with each other (Ohta et al., 1999) and depending on the it triggers the metaphase to anaphase transition and the exit E2 enzyme, Apc11 alone or together with Apc2 could support from mitosis (Harper et al., 2002). the nonspecific transfer of ubiquitin to protein substrates The APC/C has been purified and analysed biochemically (Gmachl et al., 2000; Leverson et al., 2000; Tang et al., 2001) from yeast, Xenopus and clam egg extracts and from human in vitro. Furthermore, Apc10 was suggested as the processivity cells. It turned out to be a large protein complex containing at factor for the APC/C (Carroll and Morgan, 2002). least 13 stably associated core subunits in yeasts (Passmore, Much less is known about the function of other APC/C 2004). Orthologues of ten of the yeast subunits could be subunits, such as Apc1, Apc4 and Apc5. The ida gene coding TPR subunits of the APC/C have different functions 3239 for the Drosophila homologue of Apc5 has been cloned and X chromosome. EST sequences (LD05103 and LD39177) characterised. Mutant alleles of ida show a characteristic corresponding to this gene encode a 595 amino acid TPR- mitotic phenotype suggesting that this subunit controls some containing protein of approximately 68 kDa. Extensive sub-functions of the APC/C (Bentley et al., 2002). sequence comparisons, using BLAST, gapped BLAST and The Apc3 (also known as Cdc27 or Mks; hereafter referred PSI-BLAST programs (Altschul et al., 1997), showed 30% to as Apc3/Cdc27/mks), Apc6 (also known as Cdc16; referred identity and 51% similarity of the predicted protein along its to as Apc6/Cdc16), and the Apc8 (also known as Cdc23; referred entire length to the Apc7 subunit of the human APC/C (Yu et to as Apc8/Cdc23) subunits constitute a group of structurally al., 1998). Interestingly, predicted proteins from Arabidopsis related proteins within the APC/C, all of which contain nine to (accession no. NP 850309) and Apis mellifera (accession no. ten copies of the tetratrico-peptide repeat (TPR) motifs in XP 396165) came up with very similar topology to both the tandem arrays. The TPRs are repeats of 34 amino acid structural human and Drosophila proteins. In each of these proteins, the motifs with a consensus sequence restricted only to eight number, location and the distribution of the TPR motifs were residues. There is no invariant residue even within the consensus very similar: they all contained eight TPR motifs in a tandem but amino acids at these positions are conserved in terms of size, array at their C terminus and two single repeats toward their hydrophobicity and spacing (Lamb et al., 1995; Blatch and N-terminal end (Fig. 1A). Comparison of the ten TPR motifs Lässle, 1999). The first X-ray structure of the TPR containing of the CG14444 gene product to each other showed similarity protein phosphatase-5 revealed that each motif forms two ␣- mostly confined to the consensus sequence of this motif (Fig. helices in an antiparallel, helix-turn-helix configuration (Das et 1B). However, sequence comparison of individual motifs to al., 1998). The neighbouring motifs are packed in a parallel positionally equivalent TPR motifs of related proteins from fashion resulting in the formation of a superhelical structure. other species revealed that the conservation among these TPR motifs are present in functionally divergent proteins and repeats extends outside of the consensus (Fig. 1C), suggesting thought to mediate protein-protein interactions and the assembly functional correspondence. The similarity pattern outside the of multiprotein complexes. It is believed that the TPR subunits consensus is specific to individual TPRs. Thus these of the APC/C form a scaffold-like structure that facilitates the observations suggest that the CG14444 gene encodes a putative binding of other catalytic subunits, regulatory proteins and Apc7 protein in Drosophila melanogaster. Likewise, Apc7 substrates (Passmore et al., 2005). TPR subunits are essential for orthologues appear to exist in Apis mellifera and Arabidopsis viability in yeasts, and their mutant alleles result in uniform, thaliana as well, suggesting that this protein is present in all mitotic G2-M arrest phenotype (Lamb et al., 1994). multicellular eukaryotes. Owing to the high degree of In Drosophila, the genes coding for three TPR subunits, structural and sequential correspondence between CG14444 Apc3/Cdc27/mks, Apc6/Cdc16 and Apc8/Cdc23 have previously and the genuine vertebrate Apc7, we designate the Drosophila been identified (Deak et al., 2003; Harper et al., 2002; Huang gene as Apc7 in the rest of this paper. and Raff, 2002). Characterisation of Apc3/Cdc27/mks mutants indicated that this subunit is essential for APC/C function and Apc6/Cdc16- and Apc8/Cdc23-specific dsRNA required for the degradation of both cyclin A and cyclin B (Deak expression causes larval-pupal polyphasic lethality Journal of Cell Science et al., 2003). In another study, GFP-tagged Apc3/Cdc27/Mks The inducible transgenic RNAi approach summarized in Fig. and Apc6/Cdc16 subunits localised differentially in living 2A was applied to knock down the expression of the syncytial embryos. Individual depletion of these subunits in Apc6/Cdc16, Apc7 and Apc8/Cdc23 genes. Constructs were cultured Drosophila S2 cells by
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