Department of Microbiology and Medical Zoology, Medical Sciences Campus, University of Puerto Rico, PO Box 365067, San Juan, PR 00936-5067, USA.
BMC Microbiol. 2010 Dec 9;10:317. doi: 10.1186/1471-2180-10-317.
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. 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.
Using 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.
This 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.
重要的生物过程需要在信号级联的各个层面上进行选择性和有序的蛋白质-蛋白质相互作用。G 蛋白是一族异三聚体 GTP 酶，通过将细胞表面受体与细胞质效应蛋白偶联，影响真核信号转导。通过基因敲除研究，它们与许多真菌的生长和致病性有关。在致病性二相真菌 Sporothrix schenckii 中，我们之前鉴定了一种百日咳敏感的 G 阿尔法亚基 SSG-1。在这项工作中，我们探讨了它与其他蛋白质的相互作用。
使用酵母双杂交技术，我们鉴定了 SSG-1 与其他重要细胞蛋白之间的蛋白质-蛋白质相互作用。使用共免疫沉淀法验证了这些相互作用。使用这些技术，我们鉴定了一种铁/锰超氧化物歧化酶（SOD）、一种甘油醛-3-P 脱氢酶（GAPDH）和两种离子转运蛋白，一种属于主要易化因子超家族（MFS）的铁载体-铁转运体和一种二价阳离子转运体 Nramp（天然抗性相关巨噬细胞蛋白）家族与 SSG-1 相互作用。这些蛋白的 cDNA 被测序，并进行了生物信息大分子序列分析，以进行正确的分类和功能分配。
这项研究首次报道了真菌 G 阿尔法抑制亚基与 SOD、GAPDH 和两种金属离子转运蛋白的相互作用。在这种真菌中，如此重要的蛋白作为 G 阿尔法亚基的伴侣被鉴定出来，这表明了这种 G 蛋白可能通过何种机制影响致病性和在环境压力或人类宿主内的生存能力。本研究首次报道了 S. schenckii 中的两种离子转运蛋白，并首次发现它们与真菌 G 蛋白α亚基相互作用。G 蛋白α亚基与转运分子的结合，加强了 G 蛋白在对外界信号做出反应中的作用，也突出了真菌 G 蛋白α亚基在 S. schenckii 中对营养物质感应的参与。这些相互作用表明，这些渗透酶可能在真菌中作为 G 蛋白的转受体发挥作用。
