Abstract
BackgroundImportant biological processes require selective and orderly protein-protein interactions at every level of the signalling cascades. G proteins are a family of heterotrimeric GTPases that effect eukaryotic signal transduction through the coupling of cell surface receptors to cytoplasmic effector proteins. They have been associated with growth and pathogenicity in many fungi through gene knock-out studies. In Sporothrix schenckii, a pathogenic, dimorphic fungus, we previously identified a pertussis sensitive G alpha subunit, SSG-1. In this work we inquire into its interactions with other proteins.ResultsUsing the yeast two-hybrid technique, we identified protein-protein interactions between SSG-1 and other important cellular proteins. The interactions were corroborated using co-immuneprecipitation. Using these techniques we identified a Fe/Mn superoxide dismutase (SOD), a glyceraldehyde-3-P dehydrogenase (GAPDH) and two ion transport proteins, a siderophore-iron transporter belonging to the Major Facilitator Superfamily (MFS) and a divalent-cation transporter of the Nramp (natural resistance-associated macrophage protein) family as interacting with SSG-1. The cDNA's encoding these proteins were sequenced and bioinformatic macromolecular sequence analyses were used for the correct classification and functional assignment.ConclusionsThis study constitutes the first report of the interaction of a fungal G alpha inhibitory subunit with SOD, GAPDH, and two metal ion transporters. The identification of such important proteins as partners of a G alpha subunit in this fungus suggests possible mechanisms through which this G protein can affect pathogenicity and survival under conditions of environmental stress or inside the human host. The two ion transporters identified in this work are the first to be reported in S. schenckii and the first time they are identified as interacting with fungal G protein alpha subunits. The association of G protein alpha subunits to transport molecules reinforces the role of G proteins in the response to environmental signals and also highlights the involvement of fungal G protein alpha subunits in nutrient sensing in S. schenckii. These interactions suggest that these permeases could function as transceptors for G proteins in fungi.
Highlights
Important biological processes require selective and orderly protein-protein interactions at every level of the signalling cascades
More than 60 inserts from colonies growing in quadruple drop out medium (QDO) (SD/-Ade/-His/-Leu/-Trp/ X-a-gal) from two different S. schenckii yeast cDNA libraries were analyzed for the presence of SSG-1 interacting proteins
These inserts encoded the C-terminal domains of a mitochondrial superoxide dismutase (SOD), a cation transporter of the Nramp family, a sidereophore-iron transporter and glyceraldehyde-3-P dehydrogenase (GAPDH)
Summary
Important biological processes require selective and orderly protein-protein interactions at every level of the signalling cascades. G proteins are a family of heterotrimeric GTPases that effect eukaryotic signal transduction through the coupling of cell surface receptors to cytoplasmic effector proteins. They have been associated with growth and pathogenicity in many fungi through gene knock-out studies. The heterotrimeric G proteins constitute a family of GTPases that transmit messages received at cell surface receptors (GPCR) to cytoplasmic effector proteins inside the cell [5]. The Ga-GTP and/or Gbg subunits interact with effector proteins such as enzymes or ion channels, resulting in the regulation of a broad range of cellular processes and pathways [6,7,8,9,10]. Ga subunits similar to the traditional Ga class rather than to the GPA group have been described in the filamentous fungi and plant pathogens such as Aspergillus nidulans [18], Neurospora crassa [19,20,21], Cryphonectria parasitica [22,23], and Magnaporthe grisea [24]
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