Cloning of a Cryptococcus neoformans gene, GPA1, encoding a G-protein alpha-subunit homolog

Abstract

We have isolated a gene, GPA1, from Cryptococcus neoformans by the PCR technique. DNA sequencing of the GPA1 clone suggested that it encodes a protein homologous to the G-protein alpha-subunit family. Comparison of the deduced amino acid sequence of the GPA1-encoded protein revealed that it is about 45% identical to several mammalian Gi alpha subunits and 48% identical to the G alpha protein Gpa2 from Saccharomyces cerevisiae. G alpha proteins are known to be involved in mating of other yeasts, such as S. cerevisiae and Schizosaccharomyces pombe. Southern analysis demonstrated that GPA1 is present in a single copy within the Cryptococcus genome. Isolation of the cDNA for GPA1 confirmed that the gene contains six introns within the coding region. The GPA1 transcript was identified by Northern (RNA) analysis as a 1.6-kb RNA present in exponentially growing cells of both the alpha and a mating types. Moreover, the abundance of this transcript increased in cells shifted to starvation medium. Coincubation of alpha and a cells on starvation medium is required for mating of cryptococcal cells. Thus, our results are consistent with the involvement of C. neoformans GPA1 in mating.

Conserved cAMP signaling cascades regulate fungal development and virulence

“…Both the MAP kinase and cAMP signaling cascades regulate mating and pathogenicity of C. neoformans ( [115,116], reviewed in [117,118]). The C. neoformans GK subunit Gpa1 was found to be homologous in sequence to the S. cerevisiae GK protein Gpa2 involved in nutrient sensing during pseudohyphal di¡erentiation [10,28,119]. Gpa1 plays a key role in cAMP signaling and virulence [115].…”

“…As with gpa3, uac1, and adr1 mutants of U. maydis, a ¢l1 mutant of its close relative Ustilago hordei, is also constitutively ¢lamentous and is restored to budding growth by exogenous cAMP [147,148]. Fil1 shares a high degree of sequence identity to the GK subunit Gpa1 from the basidiomycete C. neoformans, required for virulence of this human pathogen [115,119]. Like cAMP, overexpression of FIL1 also suppressed the ¢lamentous growth of starved haploid wild-type strains, suggesting that Fil1 functions to activate adenylyl cyclase [148].…”

The Cryptococcus neoformans STE11α gene is similar to other fungal mitogen‐activated protein kinase kinase kinase (MAPKKK) genes but is mating type specific

“…Preliminary molecular studies of mating in C. neoformans have identified a number of genes bearing substantial sequence similarity to S. cerevisiae mating-associated genes. These genes include homologues of GPA1 (Tolkacheva et al, 1994;Alspaugh et al, 1997), STE12 (Wickes et al, 1997), STE4 (Wang et al, 2000) and a pheromone (Moore and Edman, 1993). Various genetic and molecular manipulations of these genes have demonstrated that at least some of their functions are conserved.…”

The pheromone response pathway ofKluyveromyces lactis

“…Because some studies have indicated direct interaction of ScGpa1p with the mating-specific MAPK Ste50p (Metodiev et al, 2002) and direct coupling of the activated Ga subunit with Scp160 (an RNA-binding protein) (Guo et al, 2003), a positive role of ScGpa1p in mating cannot be discarded. Ga subunits playing positive roles in mating have been described in other fungal species, for example Gpa1p (homologous to ScGpa2p, which is involved in cAMP regulation) in the basidiomycete Cryptococcus neoformans (Tolkacheva et al, 1994;Alspaugh et al, 1997), Gpa3p in Ustilago maydis (Regenfelder et al, 1996), and Gpa1p in the fission yeast Schizosaccharomyces pombe (Obara et al, 1991). In addition to the pheromone response pathway, in K. lactis a cAMP-nutrient-sensing signalling pathway regulated by a Ga2 (KlGpa2p) subunit has been identified (Saviñón-Tejeda et al, 1996), which is unrelated to the mating pathway.…”