Phospho-regulation of Lrg1 controls polarised growth in the human fungal pathogen Candida albicans

The small GTPase Rho1 is the positive regulatory subunit of β-1,3 glucan synthase, responsible for the main structural component of the cell wall. In turn Rho1 is negatively regulated by Lrg1, its GTPase activating protein (GAP). Here it is shown that a C. albicans lrg1ΔΔ mutant constitutively forms highly elongated and invasive pseudohyphae. Using Rho1 and Exo84 as markers, it is shown that the extended polarised growth is in part due to a failure to relocate the polarity machinery from the bud tip to the bud neck. A GFP reporter that binds specifically to active Rho1 also shows this increase in polarised growth in lrg1ΔΔ is due to an increase in Rho1 activity. CaLrg1 contains 4 complete motifs and an additional 15 minimal sites for phosphorylation by Cdc28/Cdk1. These sites are clustered in an N-terminal extension missing from the S. cerevisiae Lrg1 homologue. Consistent with this, GST-Lrg1 is phosphorylated in vitro by Cdc28. Substitution of the putative Cdk1 targets with non-phosphorylatable alanine has little phenotypic effect. However, phosphomimetic glutamate substitutions results in highly polarised growth similar to, but milder than the lrg1ΔΔ phenotype. CaLrg1 also contains 4 motifs for phosphorylation by the Cbk1 kinase, one of which is also within the N-terminal extension. Cbk1 phosphorylates GST-Lrg1 in vitro and replacement of the Cbk1 motifs with phospho-mimetic residues also produces a similar phenotype to the lrg1ΔΔ strain. Phosphomimetic mutations of Lrg1 in either Cdc28 or Cbk1 sites was also shown to increase the susceptibility of C. albicans to the echinocandin class of drug, a major tool in the treatment of Candida albicans infection. Taken together these results suggest that the regulation of polarised growth of the yeast bud is mediated by Rho1 whose activity and mobility is in turn controlled by the action of both Cdc28 and Cbk1 on Lrg1