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

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.

Quantitative Proteomic Analysis in Candida albicans Using SILAC-Based Mass Spectrometry.

Stable isotope labelling by amino acids in cell culture (SILAC) in conjunction with MS analysis is a sensitive and reliable technique for quantifying relative differences in protein abundance and posttranslational modifications between cell populations. We develop and utilise SILAC-MS workflows for quantitative proteomics in the fungal pathogen Candida albicans. Arginine metabolism provides important cues for escaping host defences during pathogenesis, which limits the use of auxotrophs in Candida research. Our strategy eliminates the need for engineering arginine auxotrophs for SILAC experiments and allows the use of ARG4 as selectable marker during strain construction. Cells that are auxotrophic for lysine are successfully labelled with both lysine and arginine stable isotopes. We find that prototrophic C. albicans preferentially uses exogenous arginine and down-regulates internal production, which allow it to achieve high incorporation rates. However, similar to other yeast, C. albicans is able to metabolise heavy arginine to heavy proline, which compromised the accuracy of protein quantification. A computational method is developed to correct for the incorporation of heavy proline. In addition, we utilise the developed SILAC labelling in C. albicans for the global quantitative proteomic analysis of a strain expressing a phosphatase-dead mutant Cdc14PD .

The spatial distribution of the exocyst and actin cortical patches is sufficient to organize hyphal tip growth.

In the hyphal tip of Candida albicans we have made detailed quantitative measurements of (i) exocyst components, (ii) Rho1, the regulatory subunit of (1,3)-ß-glucan synthase, (iii) Rom2, the specialized guanine-nucleotide exchange factor (GEF) of Rho1, and (iv) actin cortical patches, the sites of endocytosis. We use the resulting data to construct and test a quantitative 3-dimensional model of fungal hyphal growth based on the proposition that vesicles fuse with the hyphal tip at a rate determined by the local density of exocyst components. Enzymes such as (1,3)-ß-glucan synthase thus embedded in the plasma membrane continue to synthesize the cell wall until they are removed by endocytosis. The model successfully predicts the shape and dimensions of the hyphae, provided that endocytosis acts to remove cell wall-synthesizing enzymes at the subapical bands of actin patches. Moreover, a key prediction of the model is that the distribution of the synthase is substantially broader than the area occupied by the exocyst. This prediction is borne out by our quantitative measurements. Thus, although the model highlights detailed issues that require further investigation, in general terms the pattern of tip growth of fungal hyphae can be satisfactorily explained by a simple but quantitative model rooted within the known molecular processes of polarized growth. Moreover, the methodology can be readily adapted to model other forms of polarized growth, such as that which occurs in plant pollen tubes.