Regulation of the anaphase-promoting complex/cyclosome by bimAAPC3 and proteolysis of NIMA

Abstract

Surprisingly, although highly temperature-sensitive, the bimA1(APC3) anaphase-promoting complex/cyclosome (APC/C) mutation does not cause arrest of mitotic exit. Instead, rapid inactivation of bimA1(APC3) is shown to promote repeating oscillations of chromosome condensation and decondensation, activation and inactivation of NIMA and p34(cdc2) kinases, and accumulation and degradation of NIMA, which all coordinately cycle multiple times without causing nuclear division. These bimA1(APC3)-induced cell cycle oscillations require active NIMA, because a nimA5 + bimA1(APC3) double mutant arrests in a mitotic state with very high p34(cdc2) H1 kinase activity. NIMA protein instability during S phase and G2 was also found to be controlled by the APC/C. The bimA1(APC3) mutation therefore first inactivates the APC/C but then allows its activation in a cyclic manner; these cycles depend on NIMA. We hypothesize that bimAAPC3 could be part of a cell cycle clock mechanism that is reset after inactivation of bimA1(APC3). The bimA1(APC3) mutation may also make the APC/C resistant to activation by mitotic substrates of the APC/C, such as cyclin B, Polo, and NIMA, causing mitotic delay. Once these regulators accumulate, they activate the APC/C, and cells exit from mitosis, which then allows this cycle to repeat. The data indicate that bimAAPC3 regulates the APC/C in a NIMA-dependent manner.

Figure 1

Temperature sensitivity of bimA1^APC3 and bimE7^APC1 strains. Strains containing no temperature-sensitive mutation (R153) or bimA1^APC3 (PM156) or bimE7^APC1 (SO4) were replica plated and incubated at permissive temperature (30°C) or semirestrictive temperature (35°C) for 3 d. Greater temperature sensitivity caused by bimA1^APC3 when compared with bimE7^APC1 is apparent.

Figure 2

Mitotic oscillations induced by bimA1^APC3 upon rapid temperature shift. (A) CMI of a bimA1^APC3 mutant strain after rapid temperature shift. (B) Autoradiograms of p34^cdc2 H1 and NIMA kinase activities and Western blot analysis of NIMA, NIME^cyclinB, and p34^cdc2. (C) Nuclear division and germling growth of the nimA5 + bimA1^APC3 mutant cells after rapid temperature upshift. Spores were first germinated at 32°C for 7.5 h, and, after harvesting by centrifugation, the germlings were rapidly transferred to new medium prewarmed to 42°C and incubated at 42°C for 8 h. The number of nuclei and germling length were measured before and 8 h after temperature shift.

Figure 3

nimA dependence of the mitotic oscillations induced by bimA1^APC3. (A) CMI of a nimA5 + bimA1 double mutant strain after rapid temperature shift. (B) Autoradiogram of p34^cdc2 H1 kinase activity and Western blots of NIMA, NIME^cyclinB, and p34^cdc2 after rapid temperature shift.

Figure 4

Effects of addition of nocodazole and MMS on the bimA1^APC3-induced mitotic oscillations. Nocodazole or MMS was added to bimA1^APC3 mutant cells 4 h after temperature shift and remained in the culture during the duration of the experiment. (A) CMI. (B) Autoradiograms of p34^cdc2 H1 and NIMA kinase activities and NIMA Western blot in the MMS-treated bimA1^APC3 cells.

Figure 5

APC/C-dependent stabilization of NIME^cyclinB and NIMA. (A) Cells of the wild-type (wt = R153) and bimA1^APC3 cells containing a copy of alcA-driven nimE^cyclinB were grown at 32°C to early log phase in acetate media (repressing for alcA). HU (100 mM) was added to the cells for 2 h to cause S phase arrest before rapid transfer to inducing medium (ethanol) also containing 100 mM HU but at 42°C. One hour after transfer to inducing medium at 42°C, glucose was added to repress expression from the alcA promoter. The abundance of NIME^cyclinB was determined by Western blotting. The same blots were also subsequently detected for p34^cdc2 as loading control. (B) The experimental procedures for NIMA induction and repression in HU-arrested cells were as described in A by using a strain with alcA inducible nimA. NIMA was detected by Western blot after immunoprecipitation. No general proteolysis was observed in the samples from which NIMA was isolated, and the level of p34^cdc2 was found by Western blotting to be constant. However, in addition to full-length NIMA (top band) numerous smaller NIMA degradation products were observed.

Figure 6

APC/C mutations override the nimA5 G2 arrest with very high p34^cdc2 H1 kinase activity. (A) Mutant cells, as indicated, were grown to early log phase at 32°C and shifted to 42°C at time 0. At the times indicated the CMI was determined after DAPI staining and fluorescent microscopy. p34^cdc2 H1 (B) and NIMA kinase (C) activities were assayed after immunoprecipitation with specific antibodies.

Figure 7

Targeted nimA deletion. (A) Restriction maps of the nimA locus and nimAΔ locus after targeted deletion. The PstI fragment of nimA was replaced with a PstI fragment containing the pyr4 gene. The linear genomic KpnI fragment containing the pyr4 insert was used for transformation in a wild-type (wt = GR5) and a bimE7^APC1 strain. Diagnostic XhoI fragments of 4.0 kb (wt) and 4.5 kb (nimAΔ) when probed with the PstI–KpnI fragment are indicated. (B) Autoradiogram of a Southern blot of XhoI-digested genomic DNA isolated from putative heterokaryons after deletion of nimA probed with the radiolabled PstI–KpnI fragment indicated in A. 10, 13, 32, 76, 109, and 131 are deletions in GR5, and 19R and 30R indicate deletions in a bimE7^APC1 strain. Heterokaryons 10, 13, 109, 19R, and 30R all contain both a specific deletion and the wild-type allele. (C) Micrographs of DAPI-stained germlings. The wild-type germling is from an 8-h sample and contains eight interphase nuclei. The nimAΔ germlings are derived from a heterokaryon germinated for 8 and 15 h, respectively, at 32°C. (D) Representative micrographs of DAPI-stained germlings from strains with the indicated pertinent genotypes. Spores were first allowed to germinate at 32°C for 7 h before shifting to 42°C for 4 h. The nimA5 mutant germling was grown at 42°C directly for 7 h. It is to be noted that the single nucleus of the large nimAΔ + bimE7^APC1 germling remained at interphase upon shift to 42°C for 4 h, whereas nuclei in the bimE7^APC1 or nimA5 + bimE7^APC1 mutant germlings all became condensed. The arrow indicates an ungerminated spore carrying the parental genotype (GR5 = pyrg89) derived from the nimAΔ + bimE7^APC1 heterokaryon.

Figure 8

Relationship between NIMA and BIMA1^APC3. To help explain why the nimA5 mutation in combination with bimA1^APC3 causes an effective mitotic arrest phenotype, whereas the bimA1^APC3 mutation generates repeating rounds of mitotic oscillation, we propose the following. (A) Substrates of the APC/C, such as cyclin B, Polo-like kinases, and NIMA, accumulate to a threshold during mitosis, which activates the APC/C to trigger their demise and also to exit from mitosis. (B) The bimA1^APC3 mutation increases the threshold of activation of the APC/C. This results in a mitotic delay during which NIMA protein and activity accumulate. When NIMA activity reaches the higher threshold level of activation, the APC/C is activated, and cells can exit mitosis. (C) In the bimA1 + nimA5 double mutant, although p34^cdc2 H1 kinase activity and NIMA5 activity are increased to allow entry into mitosis, the level of NIMA5 activity stays below that required to activate the APC/C. This then causes an extended mitotic arrest, because cells cannot exit mitosis.