ABSTRACT
The G protein α subunits Gpa1, Gpa2, and Gpa3 mediate signal transduction and are important in the growth and virulence of Cryptococcus neoformans. To understand how Gpa1 functions without a conventional Gß subunit, we characterized a resistance to inhibitors of cholinesterase 8 (Ric8) homolog from C. neoformans, which shares amino acid sequence homology with other Ric8 proteins that exhibit guanine nucleotide exchange factor (GEF) activity toward Gα. We found that the ric8 mutant was reduced in capsule size and melanin formation, which could be suppressed by cyclic AMP (cAMP) supplementation or by introducing the activated GPA1(Q284L) allele. Consistent with the fact that Ric8 participates in cAMP signaling to regulate virulence, the ric8 mutant was attenuated in virulence toward mice. Interestingly, disruption of RIC8 also resulted in opposing effects on pheromone signaling, as the ric8 mutant showed reduced mating but an enhanced ability to induce the pheromone response in the mating partner. To identify Ric8 functional mechanisms, we examined the interactions between Ric8 and the three Gα proteins. Ric8 interacted with Gpa1 and Gpa2, but not Gpa3. The presence of Gpa1(Q284L) negatively affected its interaction with Ric8, whereas the activated Gpa2(Q203L) allele abolished the interaction. Collectively, these findings suggest that Ric8 functions as a GEF to facilitate the activation of Gpa1-cAMP signaling and to promote Gpa2, affecting mating efficiency. Our study highlights the distinct and conserved characteristics associated with G protein signaling and contributes to our overall understanding of how G protein α subunits function with or without a canonical Gß partner in C. neoformans.
Subject(s)
Cholinesterases/genetics , Cryptococcus neoformans/genetics , Fungal Proteins/genetics , GTP-Binding Protein alpha Subunits/genetics , Pheromones/metabolism , Animals , Cholinesterase Inhibitors/pharmacology , Cholinesterases/metabolism , Cyclic AMP/genetics , Cyclic AMP/metabolism , GTP-Binding Protein alpha Subunits/metabolism , Mice , Pheromones/genetics , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
Gß-like/RACK1 functions as a key mediator of various pathways and contributes to numerous cellular functions in eukaryotic organisms. In the pathogenic fungus Cryptococcus neoformans, noncanonical Gß Gib2 promotes cAMP signaling in cells lacking normal Gpa1 function while displaying versatility in interactions with Gα Gpa1, protein kinase Pkc1, and endocytic intersectin Cin1. To elucidate the Gib2 functional mechanism(s), we demonstrate that Gib2 is required for normal growth and virulence. We show that Gib2 directly binds to Gpa1 and Gγ Gpg1/Gpg2 and that it interacts with phosphodiesterase Pde2 and monomeric GTPase Ras1. Pde2 remains functionally dispensable, but Ras1 is found to associate with adenylyl cyclase Cac1 through the conserved Ras association domain. In addition, the ras1 mutant exhibits normal capsule formation, whereas the ras1 gpa1 mutant displays enhanced capsule formation, and the ras1 gpa1 cac1 mutant is acapsular. Collectively, these findings suggest that Gib2 promotes cAMP levels by relieving an inhibitory function of Ras1 on Cac1 in the absence of Gpa1. In addition, using GST affinity purification combined with mass spectrometry, we identified 47 additional proteins that interact with Gib2. These proteins have putative functions ranging from signal transduction, energy generation, metabolism, and stress response to ribosomal function. After establishing and validating a protein-protein interactive network, we believe Gib2 to be a key adaptor/scaffolding protein that drives the formation of various protein complexes required for growth and virulence. Our study reveals Gib2 as an essential component in deciphering the complexity of regulatory networks that control growth and virulence in C. neoformans.
Subject(s)
Adenylyl Cyclases/metabolism , Cryptococcus neoformans/metabolism , Cyclic AMP/metabolism , Fungal Proteins/metabolism , GTP-Binding Protein beta Subunits/metabolism , ras Proteins/metabolism , Adenylyl Cyclases/genetics , Amino Acid Sequence , Blotting, Western , Cryptococcus neoformans/genetics , Cryptococcus neoformans/growth & development , Fungal Proteins/genetics , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein beta Subunits/classification , GTP-Binding Protein beta Subunits/genetics , GTP-Binding Protein gamma Subunits/genetics , GTP-Binding Protein gamma Subunits/metabolism , Mass Spectrometry , Molecular Sequence Data , Mutation , Phylogeny , Protein Binding , Protein Interaction Maps , Sequence Homology, Amino Acid , Signal Transduction/genetics , Two-Hybrid System Techniques , ras Proteins/geneticsABSTRACT
The collection of gene deletion mutants of Saccharomyces cerevisiae was used to screen for novel genes required for UV-induced mutagenesis. We found the SBF transcription factor (Swi4/Swi6 protein complex) to be required for wild-type levels of UV mutability in forward and reverse mutation assays. Expression of translesion polymerase zeta component Rev7 was identified as a target of SBF-dependent regulation.
