The anaphase-promoting complex/cyclosome is required for anaphase progression in multinucleated Ashbya gossypii cells

Abstract

Regulated protein degradation is essential for eukaryotic cell cycle progression. The anaphase-promoting complex/cyclosome (APC/C) is responsible for the protein destruction required for the initiation of anaphase and the exit from mitosis, including the degradation of securin and B-type cyclins. We initiated a study of the APC/C in the multinucleated, filamentous ascomycete Ashbya gossypii to understand the mechanisms underlying the asynchronous mitosis observed in these cells. These experiments were motivated by previous work which demonstrated that the mitotic cyclin AgClb1/2p persists through anaphase, suggesting that the APC/C may not be required for the division cycle in A. gossypii. We have now found that the predicted APC/C components AgCdc23p and AgDoc1p and the targeting factors AgCdc20p and AgCdh1p are essential for growth and nuclear division. Mutants lacking any of these factors arrest as germlings with nuclei blocked in mitosis. A likely substrate of the APC/C is the securin homologue AgPds1p, which is present in all nuclei in hyphae except those in anaphase. The destruction box sequence of AgPds1p is required for this timed disappearance. To investigate how the APC/C may function to degrade AgPds1p in only the subset of anaphase nuclei, we localized components and targeting subunits of the APC/C. Remarkably, AgCdc23p, AgDoc1p, and AgCdc16p were found in all nuclei in all cell cycle stages, as were the APC/C targeting factors AgCdc20p and AgCdh1p. These data suggest that the AgAPC/C may be constitutively active across the cell cycle and that proteolysis in these multinucleated cells may be regulated at the level of substrates rather than by the APC/C itself.

FIG. 1.

Comparison of homologous domains and sequence features in APC/C cofactors from A. gossypii and S. cerevisiae. (A) AgCdc20p (AFL014C) and ScCdc20p (YGL116W) are 59% identical on the amino acid level. The positions of a D box, a C box, and seven WD repeats are shown for each protein, and notably, a stretch of 15 identical amino acids (aa) precedes the WD repeats. Asterisks mark potential CDK phosphorylation sites (S20, S136, and S493 in AgCdc20p and S24, S173, S439, and S534 in ScCdc20p). These sites are conserved in eight yeast species and A. gossypii except S439 (in the fifth WD repeat). In all nine Cdc20p orthologues, the last amino acid is arginine preceded by two hydrophobic amino acids. (B) AgCdh1p (AFL007C) and ScCdh1p (YGL003C) are 66% identical, with the highest homology in the WD repeat region and the preceding 21 amino acids. All nine analyzed Cdh1 orthologues terminate with an arginine preceded by a hydrophobic amino acid. The eight asterisks in AgCdh1p mark CDK phosphoryation consensus sites (T12, S16, S44, T144, T161, S212, S224, and S421), all of which are conserved in eight yeast orthologues. In ScCdh1p, the homologous sites are phosphorylated with the possible exceptions of S42 and S227 (16a, 57). The three additional consensus CDK sites in ScCdh1p (S169, T173, and S418) are most likely not phosphorylated. From the 11 non-CDK phosphorylation sites identified in ScCdh1p by those authors, only 5 (S38, S172, S193, S225, and S556) are conserved in A. gossypii and other yeast Cdh1 sequences. LIR, LR, and IR refer to amino acids.

FIG. 2.

Cells lacking AgCDC23 and AgDOC1 are inviable and arrest in mitosis. (A) Spores from wild-type (WT) A. gossypii cells were grown for 15 h at 30°C and processed for antitubulin immunofluorescence and DNA staining. (B) Brightfield images depicting “late lethal” arrest point as small mycelia from Agdoc1Δ and Agcdc23Δ mutants. (C and D) Spores from Agcdc23Δ heterokaryons (RNSN01) and Agdoc1Δ heterokaryons (RNSN02), respectively, were grown under selection for 15 h at 30°C and processed for antitubulin immunofluorescence and DNA staining. Arrows point to examples of mitotic nuclei. Bar, 10 μm.

FIG. 3.

Cells lacking AgCDC20 or AgCDH1 are inviable and arrest in mitosis. (A) Brightfield images depicting arrest as uni- or bipolar germlings for Agcdc20Δ and small mycelia for Agcdh1Δ. (B) Spores from Agcdc20Δ heterokaryons (NSG01) were incubated under selection for 15 h at 30°C and processed for tubulin and DNA staining. After this incubation time, single mitotic spindles are still present in the arrested germlings but nuclei appear fragmented. (C) Spores from Agcdh1Δ heterokaryons (NSG02) were incubated under selection for 15 h prior to tubulin and DNA staining. The arrow points to an example of a mitotic spindle. Bar, 10 μm.

FIG. 4.

(A) Comparison between AgPds1p (AGR083W) and ScPds1p (YDR113C). The percent identity between the two proteins is 29%. N-terminal protein destruction motifs (KEN box and D box) are noted in addition to three conserved regions in the middle of Pds1p, one highly enriched in negatively charged amino acids (aa) and one enriched for proline, all of which are conserved in yeast species. A major difference between Pds1p orthologues of Saccharomyces species and A. gossypii is the absence of 39 amino acids upstream and 20 amino acids downstream of the D box. Pds1 orthologues from K. waltii and K. lactis also lack blocks of amino acids of similar sizes in this region. The asterisks in ScPds1p mark the positions of five CDK consensus phosphorylation sites (T27, S71, S277, S292, and T304); only two (T27 and S292) are conserved in AgPds1p (T23 and S213) and in seven other yeast orthologues. (B) Spores from Agpds1Δ heterokaryons (NSG03) were incubated under selection for 18 h at 30°C and processed for DNA and tubulin staining. Cells arrested as germlings with one germ tube, most of which carried one nucleus, arrested in either metaphase (Meta) or anaphase (Ana) (arrows denote different stage nuclei). Bar, 10 μm.

FIG. 5.

AgPds1p is present in nuclei but diminishes in anaphase. Spores from a strain expressing AgPds1p-HA (NSG09) were grown under selective conditions and then processed for immunofluorescence. (A) Segment of a branched hypha showing four nonmitotic nuclei and one anaphase nucleus (arrow). Only the nonmitotic nuclei contain AgPds1p-HA. (B) AgPds1p-HA localization in different nuclear cycle stages showing its presence in nuclei with one or two spindle pole bodies and in metaphase (Meta) nuclei but not in anaphase (Ana) nuclei. (C) Hyphae were released from a nocodazole block to follow AgPds1p-HA through anaphase in a large number of nuclei. Weak AgPds1p-HA signals are observed only in maximally extended spindles (bottom panels). Bar, 5 μm.

FIG. 6.

AgPds1Δdbp persists in anaphase nuclei. Hyphae containing a plasmid expressing AgPds1Δdb-6HA (NSG10) were scraped from a selective plate, vortexed in liquid medium to break apart the mycelia, grown under selection for 15 h, and processed for tubulin and HA epitope staining. The arrow highlights one anaphase nucleus with nondegraded AgPds1Δdb-6HA. Bar, 10 μm.

FIG. 7.

The predicted A. gossypii APC/C subunits AgCdc23p, AgDoc1p, and AgCdc16p localize to nuclei in all stages of the nuclear division cycle. (A) AgCdc23p-13myc (GVS4), (B) AgDoc1p-9myc (NSG11), and (C) AgCdc16p-13myc (GVS2) cells were grown for 16 h at 30°C under selective conditions and processed for anti-myc and antitubulin immunofluorescence. In AgDOC1p-13myc (GVS3), a signal was also observed for all nuclear cycle stages. Arrows highlight nuclei in different stages of the division cycle, where “1” indicates a nucleus with a single SPB and “2” indicates a nucleus with duplicated SPBs. Meta, metaphase; Ana, anaphase. Bar, 10 μm.

FIG. 8.

APC activators AgCdh1p and AgCdc20p localize to all nuclei independent of the nuclear cycle stage. (A) Immunostained hyphae expressing N-terminally GFP-labeled AgCdh1p (NSG12). (B) Immunostained hyphae expressing N-terminally GFP-labeled AgCdc20p (NSG13). All nuclei, including anaphase nuclei (arrows), show anti-GFP immunofluorescence. The nuclei of nontransformed hyphae were not stained (see Fig. S1 in the supplemental material). Bar, 10 μm.