Proteins that physically interact with the phosphatase Cdc14 in Candida albicans have diverse roles in the cell cycle

Abstract

The chromosome complement of the human fungal pathogen Candida albicans is unusually unstable, suggesting that the process of nuclear division is error prone. The Cdc14 phosphatase plays a key role in organising the intricate choreography of mitosis and cell division. In order to understand the role of Cdc14 in C. albicans we used quantitative proteomics to identify proteins that physically interact with Cdc14. To distinguish genuine Cdc14-interactors from proteins that bound non-specifically to the affinity matrix, we used a substrate trapping mutant combined with mass spectrometry analysis using Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC). The results identified 126 proteins that interact with Cdc14 of which 80% have not previously been identified as Cdc14 interactors in C. albicans or S. cerevisiae. In this set, 55 proteins are known from previous research in S. cerevisiae and S. pombe to play roles in the cell cycle, regulating the attachment of the mitotic spindle to kinetochores, mitotic exit, cytokinesis, licensing of DNA replication by re-activating pre-replication complexes, and DNA repair. Five Cdc14-interacting proteins with previously unknown functions localised to the Spindle Pole Bodies (SPBs). Thus, we have greatly increased the number of proteins that physically interact with Cdc14 in C. albicans.

Figure 1

Characterisation of Cdc14^PD. (a) Time course of Cdc14^PD-Myc expression. C. albicans cells were grown overnight and then starved in water for 4 hours to induce transition into G0. They were then released into fresh medium and left to grow in either yeast- or hyphae-promoting conditions. Aliquots were removed every 15 min and processed for Western blotting using an anti-Myc monoclonal antibody. An anti-PSTIARE antibody that recognises Cdk1 and Pho85 was used as a loading control. The phosphatase is not present in stationary phase cells and it starts appearing after about 45 min in yeast and 60 min in hyphae. The temporal pattern of Cdc14-Myc and Cdc14^pd-Myc expression were similar. Both Cdc14-Myc and Cdc14^PD-Myc are expressed at lower levels in hyphae. Note the presence of a retarded band in Cdc14^PD-Myc showing increased phosphorylation compared to Cdc14-Myc. (b) Treatment of the lysate from C. albicans cells expressing Cdc14^PD-Myc with λ phosphatase results in the disappearance of the retarded band showing the protein is phosphorylated. Full length autoradiograms are presented in Supplementary Fig. S1. (c) Cdc14^PD-Myc immunoprecipitates Cdc14-GFP showing a physical interaction. Cell lysates were immunoprecipitated with αMyc (IP) and the resulting Western blot probed with αMyc or αGFP as indicated.

Figure 2

Identification of Cdc14 interactors using SILAC. (a) Experimental strategy. CDC14-/ MET3-cdc14^PD–Myc cells were grown in depressing heavy medium alongside parental untagged cultures grown in light medium and the lysates mixed in equal quantities prior to mass spectrometry analysis. Cdc14^PD–Myc was immunoprecipitated and the immuneprecipitate analysed using a quadrupole Orbitrap-MS. Peptides from genuine interacting proteins will show a significant deviation from 1:1 H:L ratio. (b) Volcano plot of intensity versus H:L ratio of detected proteins. Blue dots show proteins enriched in heavy peptides in both yeast and hyphae, yellow dots show proteins only enriched in heavy peptides in the yeast form and grey dots indicate proteins that show no heavy peptide enrichment. Proteins detected in the yeast IPs or hyphal IPs but not detected in the respective lysates are listed in Supplementary Dataset S2.

Figure 3

Five ORFs of previously unknown function localise to the SPB. C-terminal fusions of the indicated ORFs to GFP were constructed (Table 1). Overnight cultures of yeast cells were re-innoculated into YEPD yeast-promoting conditions and incubated for 4 hours. Cells were fixed and stained with DAPI. GFP and DAPI fluorescence was imaged using a Delta Vision RT microscope as described in materials and methods. Representative images of cells at different stages of the cell cycle are shown as maximum intensity projections of the green GFP signal and the blue DAPI signal.

Figure 4

Role of 55 CaCdc14-interacting proteins in the cell cycle. The cartoon represents the cell and nuclear cycles. Blue circles represent nuclei, black dots SPBs, chromosomes are shown above, replicating early in the cycle and then attached to MTs (blue lines) through kinetochores before sister chromatid separation at anaphase. Thick black lines show DNA damage and spindle checkpoints. After anaphase a contractile actomyosin ring forms (orange ring) which then contracts to a spot (orange) to guide the formation of the primary septum during cytokinesis before cell separation. Proteins also known to physically interact with ScCdc14 are shown in black font, other proteins are shown in blue font. Where the S. cerevisiae orthologue is shown in brackets the standard name in CGD is different, but the S. cerevisiae name is annotated as an alias or homolog in CGD. Protein functions are taken from the annotation in SGD or CGD. ORF19.7060 is included in DNA repair category as it contains and InterPro XLF domain implicated in double strand break repair (see text). CPC: Cargo passenger complex. APC: Anaphase promoting complex.

Figure 5

Cdc14 dephosphorylation motifs are enriched in the set of Cdc14 interacting proteins. (a) Box plots of the number of consensus Cdc14 dephosphorylation motifs found proteins classified according to whether they were identified in a set of Cdc14-interacting proteins (hits) in hyphae only, yeast only, both yeast and hyphae or not present in the total set of hits. Boxes show the second and third quartiles, the thick horizontal line shows the median, and the vertical line shows the extent of samples within 1.5x of the interquartile range. (b) Plots of the percentage of all proteins carrying the indicated number of target motifs that were identified in the set of hits. The shaded area shows the 95% confidence limits.