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  current news   Press   selected story    
     
  13th January 2010  
 

Discovery of novel regulatory mechanisms for Candida albicans hyphal development

 
 




Title: Candida albicans Cyr1, Cap1 and G-actin form a sensor/effector apparatus for activating cAMP synthesis in hyphal growth.

Author: Hao Zou, Hao-Ming Fang, Yong Zhu and Yue Wang*

* Corresponding author

Abstract
A key virulence trait of Candida albicans is its ability to undergo the yeast-to-hyphal growth transition in response to environmental signals. This transition critically requires a rapid activation of the adenylyl cyclase Cyr1 to generate a cAMP spike. However, the identity of the signal sensors and mechanisms of signal processing and integration remain largely unclear. Recent evidence suggests that some sensors are embedded in Cyr1 itself. To test this hypothesis, we asked whether purified Cyr1 can respond to hyphal induction. Here, we report that Cyr1 copurifies with Cap1 and G-actin as a tripartite complex which can increase cAMP synthesis in response to hyphal inducing signals in an actin-dependent manner. Cap1 binds Cyr1 and G-actin through its N and C-terminus respectively. Deleting the G-actin-binding sites or treating the complex with the actin toxin latrunculin A or cytochalasin A inhibits the activation of cAMP synthesis. Strains expressing Cap1 mutants lacking the G-actin-binding site are impaired in both cAMP synthesis and hyphal morphogenesis. Thus, our findings reveal an essentially intact sensor/effector apparatus composed of Cyr1, Cap1 and G-actin. Furthermore, G-actinís regulatory role in this apparatus may prove to be the missing link whereby cellular actin status knowingly influences cAMP-mediated cellular processes.

 
 


 
 

Figure Legend: Characterization of interactions between Cyr1, Cap1 and G-actin by co-IP.
A) Schematic description of the domain organization of Cap1 and the mutant versions of Cap1 constructed for this study. N30, N-terminal 30 aa; P1 and P2: proline-rich region 1 and 2; and C27, C-terminal 27 aa. B) A hypothetical Cyr1/Cap1/actin tripartite complex in which Cap1 acts as a bridge binding Cyr1 and G-actin through its N- and C-terminal ends respectively. Deleting either N30 or C27 is predicted to disrupt the complex. C) co-IP analysis of Cyr1, Cap1 and actin. Strains used are Cap1-WT, cap1Δ/Δ-1, Cap1-N30Δ, Cap1-P1Δ, Cap1-P1Δ and Cap1-C27Δ. Proper expression of HFM-Cyr1, the various versions of Cap1-GFP and actin in cell lysates were first verified by western blotting (top panel) using Myc (Roche), GFP (Clontech) and actin (Santa Cruz) antibodies, respectively. Then, IP was performed from all lysates using GFP (middle panel) or Myc (bottom panel) antibody, followed by western-blot analysis of HFM-Cyr1, Cap1-GFP and actin using respective antibodies.

Mol. Microbiol. (2009) Nov 25. Epub ahead of print.

 

 
 

 

Title: Ras1 and Ras2 play antagonistic roles in regulating cellular cAMP level, stationary-phase entry and stress response in Candida albicans.

Authors: Yong Zhu, Hao-Ming Fang, Yan-Ming Wang, Gui-Sheng Zeng, Xin-De Zheng and Yue Wang*

* Corresponding author


Abstract
The GTPase Ras1 activates the yeast-to-hypha transition in Candida albicans by activating cAMP synthesis. Here, we have characterized Ras2. Ras2 belongs to a group of atypical Ras proteins in some fungal species that share poor identity with other Ras GTPases with many variations in conserved motifs thought to be crucial for Ras-associated activities. We find that recombinant Ras2 is enzymatically as active as Ras1. However, only RAS1 can rescue the lethality of the Saccharomyces cerevisiae ras1 ras2 mutant, suggesting functional divergence of the two genes. ras2Δ is normal in hyphal growth, but deleting RAS2 in the ras1Δ background greatly aggravates the hyphal defect, indicating that Ras2 also has a role in hyphal development. Strikingly, while RAS1 deletion causes a ~20-fold decrease in cellular cAMP, further deletion of RAS2 restores it to ~30% of the wild-type level. Consistently, while the ras1Δ mutant enters the stationary phase prematurely, the double mutant does so normally. Moreover, ras1Δ cells exhibit increased resistance to H2O2 and higher sensitivity to the heavy metal Co2+, whereas ras2Δ cells show the opposite phenotypes. Together, our data reveal a novel regulatory mechanism by which two antagonizing Ras GTPases balance each other in regulating multiple cellular processes in C. albcians.


 
 


 

 
 

Figure Legend: Ras1 and Ras2 exhibited growth-phase-dependent expression patterns.
A) Western-blot analysis of Ras1 and Ras2 cellular levels in different phases of growth. A strain expressing GFP-Ras1 (WY-ZXD3) and one expressing both 6◊Myc-Ras2 and 6◊Myc-Cdc42 (WY-ZY15) were used. Overnight cultures were diluted into pre-warmed GMM to a density of ~1◊107 cells /ml for growth at 30įC and aliquots containing approximately the same number of cells were taken at the indicated times for protein extraction. Cell lysates expressing GFP-Ras1 were directly used for western blotting analysis using a GFP antibody and a PSTAIRE antibody. For the anti-Myc detection of Ras2 and Cdc42, membrane proteins were prepared before western blotting. B) RT-PCR quantitation of RAS1 and RAS2 mRNA levels. The mRNA levels were quantified using real-time PCR and normalized against 18S ribosome RNA. The relative mRNA levels of RAS1 and RAS2 were presented by setting the amount of mRNA detected at 6 hr as 1. The data are averages of results from two independent assays where each data point was analyzed in triplicate. C) Cellular cAMP levels were measured in cells taken at the same time point as above. Average values of three independent experiments and standard errors are shown.

Published in Mol. Microbiol. (2009) 74:862-875.

For more information on Wang Yue's Lab, please click here.