RA domain‐mediated interaction of Cdc35 with Ras1 is essential for increasing cellular cAMP level for Candida albicans hyphal development

Abstract

SummaryMany Ras GTPases activate their effectors through binding at a conserved Ras association (RA) domain. An example is the activation of the budding yeast adenylate cyclase Cyr1 by Ras1 and Ras2. Candida albicans Ras1 is speculated to similarly activate Cdc35, the orthologue of Cyr1, for hyphal development. Here, we have investigated whether the RA domain mediates Ras1-Cdc35 interaction and how this interaction regulates cAMP levels and morphogenesis. Yeast two-hybrid assays suggested that Ras1 interacts only with the RA but not any other identifiable domains of Cdc35. The Ras1-RA interaction was further confirmed by in vitro binding assays of purified RA domain and Ras1 and by co-immunoprecipitation of Ras1 and Cdc35 from cell lysates. Substituting Ala for the conserved residue K 338 or L 349 in the RA domain or deleting the RA domain abolished the Ras1-RA or Ras1-Cdc35 interactions. cdc35 mutants with the RA domain deleted or carrying K388A or L349A mutation exhibited rather normal yeast growth but were completely defective in hyphal morphogenesis. Further, the mutants contained nearly wild-type levels of cAMP during yeast growth but were unable to increase it upon hyphal induction. These results suggest an essential role for the RA-mediated Ras1-Cdc35 interaction in raising cellular cAMP levels for hyphal morphogenesis.

Environmental Sensing and Signal Transduction Pathways Regulating Morphopathogenic Determinants of Candida albicans

“…Both phenotypes are shared with mutants lacking the guanine nucleotide exchange factor Cdc25 (193), suggesting that the Cdc25-Ras1 pathway functions upstream of the cAMP-PKA pathway. This is supported by a recent study in which a clear interaction between Ras1 and adenylate cyclase, Cyr1, is demonstrated (90). However, as with the situation in S. cerevisiae, Ras1 also appears to function upstream of the Cek1 MAPK pathway ( Fig.…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…Ras1 and Gpa2 both function upstream of the adenylyl cyclase Cyr1, and the physical interaction between Ras1 and Cyr1 is essential for increasing cellular cAMP levels (173,490). Cyr1 is the single adenylyl cyclase in C. albicans and therefore is the sole source of cAMP production in the cell.…”

Identification of inhibitors of yeast-to-hyphae transition in Candida albicans by a reporter screening assay

“…The cAMP-protein kinase A-pathway plays a central role in the induction of hyphae: the adenylate cyclase Cyr1p is activated by a number of stimuli, such as CO 2 -concentration, and signals from other proteins, e.g. the small G-protein Ras1p and the cytoskeleton (Fang and Wang, 2006;Klengel et al, 2005;Rocha et al, 2001;Zou et al, 2010). Ras1p itself is activated in response to multiple signals, like nitrogen shortage or the presence of serum (Biswas and Morschhäuser, 2005;Feng et al, 1999).…”