Abstract

The G1/S transition is a critical control point for cell proliferation, and involves the essential transcription complexes SBF and MBF in Saccharomyces cerevisiae, or MBF in Schizosaccharomyces pombe. In S. cerevisiae, Swi4p and Mbp1p comprise the DNA binding elements for SBF and MBF, respectively, while Swi6p is a common activating component. In the fungal pathogen Candida albicans, G1/S regulation is not yet clear. Orthologues of Swi6p, Swi4p and Mbp1p exist and previous work suggested that Swi4p and Swi6p form a single G1/S transcription factor complex, while the function of Mbp1p remained unclear as its absence did not affect growth. Additionally, unknown factors were suggested to contribute to G1/S regulation in C. albicans as cells lacking Swi4p and Swi6p, or Swi4p and Mbp1p were still viable, unlike the situation in S. cerevisiae. A previous graduate student from the Bachewich lab demonstrated through tandem-affinity purification of Swi4p, Swi6p and Mbp1p coupled with Orbitrap LC/MS, and co-immunoprecipitation that Swi6p interacted with Swi4p as well as Mbp1p, but an interaction between Swi4p and Mbp1p was not clear, questioning the current model that Swi4p and Swi6p are the major components of a single MBF-like complex in C. albicans. Additional putative interacting proteins were identified but not validated. Further, identification of Swi4p targets using genome-wide location analysis revealed cell-cycle related factors but also regulators of filamentous growth, including EFG1. In this study, the composition of the putative G1/S transcription factor complex was further investigated using co-immunoprecipitation experiments that utilized lower amounts of input protein and variations in epitope tags. The results confirm that Swi6p similarly interacts with Swi4p and Mbp1p. However, Swi4p and Mbp1p showed a weak interaction that could only be detected with higher amounts of input protein and only when Swi4p was immune-precipitated. Thus, separate Swi6p/Swi4p and Swi6/Mbp1p complexes may exist in C. albicans, but the function of the Swi6p/Mbp1p complex remains unknown. We next carried out co-immunoprecipiation experiments to validate additional proteins identified in the previous Swi6p affinity purification screen, including the mitotic polo-like kinase Cdc5p. When Cdc5p was immune-precipitated from G1-phase arrested cells, Swi6p co-purified, suggesting a novel interaction between these two proteins. Finally, in order to validate the functional significance of Swi4p occupation of the EFG1 promoter, EFG1 expression in the presence and absence of Swi4p was investigated by Northern blotting, and the effects of deleting EFG1 on the Swi4p-depleted phenotype were determined. In the absence of Swi4p, EFG1 was moderately induced. Furthermore, absence of EFG1 reduced the extent to which swi4/swi4 cells became enlarged and formed long filaments. Thus, Swi4p may contribute to the regulation of EFG1 and possibly filamentous morphogenesis, as well as the G1/S transition, suggesting that it may lie at the interface between cell cycle regulation and development in C. albicans.