Homologous interacting protein pairs from additional organisms (interlogs) were not found. Open in a separate window Figure 1 Arabidopsis heterotrimeric G-protein core interactome. high-quality G-protein interactome map using candida two-hybrid screening Aiming at the recognition of regulators and effectors of heterotrimeric G-protein subunits in cells were screened 10 instances using seven main baits, including the subunits of the heterotrimeric G-proteins G (GPA1), G/G1 (AGB1/AGG1), G/G2 (AGB1/AGG2), (Ullah et al, 2001, 2003; Trusov et al, 2007), and regulator of G-protein signaling 1 (RGS1), Pirin (PRN), N-myc downregulated-like1 (NDL1), and receptor for triggered C kinase1A (RACK1A; Chen et al, 2003, 2006; Lapik and Kaufman, 2003; Mudgil et al, 2009). To distinguish between potential effectors that interact with GPA1 in its active conformation only, and Teglicar regulators interacting with GPA1 irrespective of its activation state, three variants of GPA1 were used as bait constructs: a constitutive active form (Q222L), the wild-type protein, and a mutant version with accelerated GTPase activity (G220A). Potential false positives were excluded both by re-cloning and retesting positive relationships, as well as excluding known common false positives (Methods and Supplementary Experimental Methods). The complete data set is available in Supplementary Table 1, in Arabidopsis Protein Interaction Teglicar Database (AtPID, ID:FDS1176001), and in a general public database dedicated to Arabidopsis G-protein signaling (AGIdb), http://bioinfolab.unl.edu/AGIdb. In accordance with published data, our screenings recognized AGG1, AGG2, and NDL1 as interactors of AGB1, and thylakoid formation 1 (THF1) as an effector of the constitutive active form of GPA1 (Huang et al, 2006; Mudgil et al, 2009). From the total of 206 unique prey proteins from the 1st round of screenings, 14 secondary baits (ANNAT1, ARD1, CDC48B, NDL1, RACK1B, RACK1C, SYP23, TGA1, UNE16, VAP27, AT1G05000, AT1G52760, AT4G26410, and AT5G14240) were chosen, because these interacted with two or more of the primary baits. Interestingly, the connection data reveal a highly interconnected network, with many proteins identified as connection partners of two or more of the bait proteins. High local connectivity helps the close practical relationship of the proteins involved, and proteins sharing connection partners have an increased probability of interacting themselves (Milo et al, 2002; Barabasi and Oltvai, 2004). Furthermore, relationships within three- and four-protein-interaction loop motifs are considered reliable and functionally meaningful (Spirin and Mirny, 2003; Wuchty et al, 2003; Yeger-Lotem et al, 2004). Consequently, we focused on the bi-connected core (two-core) of the network composed of 69 proteins interacting with at least two additional proteins in the network. Thirty-eight of these proteins were analyzed further by separately screening all possible pair-wise mixtures for connection in candida. This approach led to the recognition of 64 additional interactions, resulting in a highly interconnected core network (68 nodes, 167 edges) with an average node degree of 4.1 (Number 1). A comparison of the gene ontology annotations of the core network to the genome indicated enrichment in these Teglicar groups: plasma membrane, nucleus, cytosol, endoplasmic reticulum, ribosome, protein binding, nucleotide binding, structural molecule activity, reactions to biotic/abiotic stresses, developmental processes and cell corporation, and biogenesis (Supplementary Number S1). Homologous interacting protein pairs from additional organisms (interlogs) were not found. Open in a separate window Number 1 Arabidopsis heterotrimeric G-protein core interactome. Only those proteins from your interactome data arranged are demonstrated that possess at least two contacts within the network (two-core). Red nodes highlight published components of G-protein Col11a1 signaling in Arabidopsis that were used as main baits in the Y2H screenings. Proteins used as baits for the second round of screens are demonstrated in brown. Gray lines (edges) represent relationships detected with the Y2H system. Green edges represent relationships demonstrated with both the Teglicar Y2H system and BiFC. Orange edges represent interactions found in our Y2H screenings that were published and confirmed by FRET analyses or co-immunoprecipitation previously (Lapik and Kaufman, 2003; Adjobo-Hermans et al, 2006; Huang et al, 2006; Mudgil et al, 2009). The AGB1-ARD1 through ARD4 confirmations are as explained in Friedman et al (2011). Solid.