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The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that coordinates progression through the cell cycle by temporally and spatially promoting the degradation of key proteins. Many of these targeted proteins have been shown to play important roles in regulating orderly progression through the cell cycle. Using a previously described Drosophila in vitro expression cloning approach, we screened for new substrates of the APC in Xenopus egg extract and identified Drosophila MCPH1 (dMCPH1), a protein encoded by the homolog of a causative gene for autosomal recessive primary microcephaly in humans. The dMCPH1-B splice form, but not the dMCPH1-C splice form, undergoes robust degradation in Xenopus interphase egg extract in a Cdh1-dependent manner. Degradation of dMCPH1-B is controlled by an N-terminal destruction box (D-box) motif as its deletion or mutation blocks dMCPH1-B degradation. dMCPH1 levels are increased in Drosophila morula (APC2) mutant embryos, consistent with dMCPH1 being an APC substrate in vivo. Using a purified, reconstituted system, we show that dMCPH1-B is ubiquitinated by APC(Cdh1), indicating that the effect of APC on dMCPH1-B ubiquitination and degradation is direct. Full-length human MCPH1 (hMCPH1) has been predicted to be an APC substrate based on its interaction with the APC subunit Cdc27. We were not able to detect changes in hMCPH1 levels during the cell cycle in cultured human cells. Overexpression of hMCPH1 (or dMCPH1-B) in developing Xenopus embryos, however, disrupts cell division, suggesting that proper regulation of hMCPH1 and dMCPH1-B activity plays a critical role in proper cell-cycle progression.
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Fig. 4. Overexpression of dMCPH1-B or hMCPH1 results in cell-cycle defects. (A) Representative images of whole Xenopus embryos fixed four hours after injection of Mos, GFP, full-length human MCPH1 (hMCPH1), dMCPH1-B, or dMCPH1-BDboxMut RNA at the 2–4-cell stage. Arrows indicate injected halves of embryos. (B) Quantification of Xenopus embryos displaying cell division defects 4 hours post-injection. Total number of embryos injected is indicated in parentheses. *p<0.005 (C) Confocal sections of the uninjected (left) and injected (right) areas of a representative whole embryo following injection with hMCPH1 mRNA. Microtubules, green; DNA, red. Scale bar: 100 µm.
Fig. S1. APC regulates stability of dMCPH1-B and dMCPH1-C. (A) Schematic representation of dMCPH1-B, dMCPH1-C, and hMCPH1. (B) Quantitation of pixel intensity of autoradiogram in Fig. 2B. Percent of initial pixel intensity was plotted over time for radiolabeled dMCPH1-B, dMCPH1- C, or Cyclin B incubated in Xenopus interphase egg extract in the absence or presence of Cdh1. **p,0.005, *p,0.05.
Fig. S2. APC does not regulate the stability of hMCPH1. (A) Autoradiogram of radiolabeled hMCPH1 incubated in Xenopus interphase egg extract in the absence or presence of Cdh1. (B) Levels of hMCPH1 do not notably fluctuate in a cell cycle-dependent manner. Immunoblot analysis of hMCPH1, Cyclin B, Cyclin A, p27, and Cdk1 in lysates derived from synchronized HeLa cells 0–20 hours after nocodazole release. Degradation of Cyclin A and B occurs in prophase and metaphase, respectively, whereas degradation of p27 marks late G1/S.
Fig. S3. Xenopus embryos express hMCPH1-Myc, dMCPH1-B-Myc, and dMCPH1-BDboxMut-Myc at similar levels. Immunoblot for Myc and tubulin (loading control) of lysates derived from Xenopus embryos after injection with mRNA encoding hMCPH1-Myc, dMCPH1-B-Myc, or dMCPH1-BDboxMut-Myc.
Confocal sections of the uninjected Xenopus embryo, NF stage 10.5 stained with Tuba4b Ab2 (DM1A, Sigma) Microtubules, green; DNA, red. Scale bar: 100 um.
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