Acm1 contributes to nuclear positioning by inhibiting Cdh1-substrate interactions

Abstract

The anaphase-promoting complex (APC) is tightly regulated during cell division, often by pseudosubstrate binding to its coactivators Cdh1 and Cdc20. Budding yeast Acm1 is a Cdh1 pseudosubstrate inhibitor whose biological function is unknown. We show here that cells lacking Acm1 have defects in nuclear positioning and spindle morphology during mitosis. However, Cdh1 substrates are not destabilized in the absence of Acm1 and expression of inactive Cdh1 mutants that retain substrate binding is sufficient for the acm1 phenotype. We conclude that Acm1 is not required to inhibit APC^Cdh1 activity but rather prevents untimely Cdh1-substrate interactions. We further provide evidence suggesting that the substrate primarily responsible for the acm1 phenotype is the bud neck-localized kinase, Hsl1. Our results imply that at least some coactivator-substrate interactions require regulation. Several unrelated APC pseudosubstrates have been identified in diverse eukaryotes and their ability to simultaneously inhibit enzymatic activity and substrate binding may partly explain why this regulatory mechanism has been selected repeatedly during evolution.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by NSF grant MCB 0841748 to M.C.H. The Purdue Research Foundation provided support to J.S.M. We thank Kerry Bloom and Jeff Moore for comments and suggestions. We thank Janet Burton and Mark Solomon for providing the cdh1-D12 and hsl1-mdb/mkb mutants and communicating results prior to publication. We thank Dr. Aaron Taylor of the Bindley Bioscience Imaging Facility for assistance with the confocal microscopy.

Figures and Tables

Figure 1 Cells lacking Acm1 have spindle defects when mitotic exit is delayed. (A) cdc15-2 ACM1 and cdc15-2 acm1Δ cells expressing GFP-Tub1 were arrested at 37°C, fixed, treated with DAPI, and the spindle and DNA observed by fluorescence microscopy. (B) Quantification of abnormal spindles from (A) as described in Methods. Data are means of three experiments with standard deviations. (C) The indicated strains (cdc15-2 background) were quantified as in (B) (n > 500 cells per strain). Centromeric plasmids expressing Acm1 variants from the ACM1 promoter were used for complementation of cdc15-2 acm1Δ (+). (D) Same as (A), comparing cdc15-2, cdc15-2 acm1Δ and cdc15-2 acm1Δ cdh1Δ. (E) Quantification of abnormal spindles from (D) (n > 370 cells per strain).

Figure 2 Cells lacking Acm1 display a nuclear position defect. (A) The indicated strains (BY4741 background) were grown at 12°C, fixed, treated with DAPI and visualized by fluorescence microscopy. Arrows show examples of binucleate mother cells (DNA segregation within the mother). (B) Quantification of binucleate mothers from (A) as described in Methods. *p < 10^-6 from chi square test compared with dyn1Δ. (C) Same as (A and B) for ACM1 and acm1Δ strains either at 12°C or in cdc15-2 background arrested at 37°C. Images are from the experiment at 12°C. Binucleate mothers (white arrow) were quantified as described in Methods. (D) dyn1Δ acm1Δ cells were complemented with a CEN plasmid expressing either ACM1, acm1-db3/ken or acm1-T161A alleles from the ACM1 promoter (+), and nuclear position was quantified as in (B). (E) Complementation of acm1Δ cells as in (D). (F) Multinucleate cells (white arrows) in the indicated strains were detected as in (A). (G) Additional examples of multinucleate dyn1Δ acm1Δ cells from (F). (H) Quantification of cells with more than two nuclei from (F). For all parts with quantitative analyses, n > 550 cells per strain. When present, error bars are standard deviations of the mean of three experiments. Asterisks (*) in (C–E and H), p < 10^-6 from chi square test compared with control (first bar).

Figure 3 Lack of Acm1 does not destabilize Cdh1 substrates. (A) Levels of Cdh1 substrates at the indicated timepoints after release of cdc15-2 ACM1 and cdc15-2 acm1Δ cultures from an α factor-induced G₁ arrest were compared by immunoblotting. The synchronized cells were released at restrictive temperature (37°C) to cause arrest in late anaphase. Note that all blots (and the anaphase quantification) are from the same experiment using ASE1-6HA strains except the Hsl1 blot, which was from an identical experiment using HSL1-9myc strains. Similar results were also obtained for Fin1-6HA (not shown). Anaphase arrest was confirmed by monitoring DNA segregation (DAPI). G6PD, loading control. (B) Same as (A), except strains with CIK1-9myc were released from S phase arrest at 37°C, because α factor arrest in G₁ results in expression of a truncated isoform that is resistant to APC.16

Figure 4 Spindle defects in acm1Δ are independent of APC^Cdh1 activity. (A) GFP-Tub1 was monitored by fluorescence microscopy in cdc15-2 acm1Δ cdh1Δ cells (cdh1Δ) complemented with integrating plasmids expressing 3FLAG-Cdh1 or 3FLAG-Cdh1-C/IR from the CDH1 promoter or a CEN plasmid expressing 3FLAG-Cdh1-WD from the ADH promoter (+) and arrested at 37°C. (B) Quantification of abnormal spindles from (A) as described in Methods. (C) Same as (A), with complementation by an integrating plasmid expressing 3FLAG-Cdh1-D12 from the CDH1 promoter. (D) Quantification of (C), including complementation by wild-type Cdh1. For (B and D), n > 400 cells per strain. *p < 10^-6 from chi square test compared with control (first bar). (E) Clb2 levels in asynchronous or G₁-arrested cells of the indicated strains used in (A and C) were monitored by immunoblot as a measure of APC^Cdh1 activity. G6PD, loading control.

Figure 5 acm1Δ phenotypes correlate with Cdh1-Hsl1 interaction and localization to the bud neck. (A) Representative fields of view showing localization of 3FLAG-Cdh1-EGFP fusion protein expressed from P_ADH1 on a CEN plasmid in cdc15-2 cdh1Δ (left) and cdc15-2 cdh1Δ acm1Δ (right) cells arrested at 37°C for 3 h prior to microscopy. Arrowheads indicate examples of bud neck localization. (B) The percentage of large-budded cells containing detectable bud neck staining from the experiment in (A) was quantified. In addition, localization was quantified after addition of a plasmid expressing ACM1 from its natural promoter (+ACM1). A minimum of 140 cells were counted per strain. *p < 0.0005 in chi square analysis compared with ACM1 (wild type, first bar). (C) Localization of wild-type Cdh1 and the Cdh1-D12 mutant as described in (A) in asynchronously growing cells and cells arrested in S phase with hydroxyurea. (D) Expression of 3FLAG-Cdh1-EGFP (wild type) and 3FLAG-Cdh1-D12-EGFP were compared in the asynchronous cultures used for (C) by immunoblot with anti-FLAG antibody. G6PD is a loading control. The negative control sample (Neg Control) is from cdc15-2 cdh1Δ acm1Δ cells without an expression plasmid and contains a faint non-specific band commonly observed in our FLAG immunoblots that co-migrates with the FLAG-Cdh1-EGFP fusion protein. (E) Representative fields of view showing localization of 3FLAG-Cdh1-EGFP expressed from P_ADH1 on a CEN plasmid in acm1Δ and acm1Δ hsl1Δ cells either growing asynchronously or arrested in S phase with hydroxyurea. Arrowheads highlight examples of bud neck localization.