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

Iliyana N Kaneva,Mark J Dickman,Ian M Sudbery,Peter E Sudbery

doi:10.1038/s41598-019-42530-1

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.

Highlights

Candida albicans is normally a harmless commensal of the skin, urogenital and gastrointestinal tracts
Cdc14PD-Myc was phosphorylated, as indicated by the band shift seen on a Western blot, which disappeared upon phosphatase treatment (Fig. 1b)
Clp[1] is subject to autodephosphorylation in S. pombe[21] and we have found CaCdc14PD-Myc physically interacts with Cdc14-GFP (Fig. 1c) suggesting this may be the case in C. albicans

Summary

Introduction

Candida albicans is normally a harmless commensal of the skin, urogenital and gastrointestinal tracts. Lacking a sexual cycle, C. albicans can undergo a parasexual cycle in which two diploid cells mate to form a tetraploid, which sheds chromosome during subsequent mitotic divisions to regain either diploid or aneuploidy states[4] Stresses, such as exposure to the antifungal drug fluconazole, result in ploidy changes, loss of heterozygosity and whole chromosome and segmental aneuploidy (reviewed in[5,6,7,8]). We have used Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC) in conjunction with quantitative MS analysis to readily distinguish physically-interacting partners against a large number of background proteins[17,18,19,20] This powerful approach has led to new insights into the involvement of Cdc[14] in the cell cycle in C. albicans

Methods

Results

Discussion

Conclusion