Identification and characterization of GIP1, an Arabidopsis thaliana protein that enhances the DNA binding affinity and reduces the oligomeric state of G-box binding factors

Abstract

Environmental control of the alcohol dehydrogenase (Adh) and other stress response genes in plants is in part brought about by transcriptional regulation involving the G-box cis-acting DNA element and bZIP G-box Binding Factors (GBFs). The mechanisms of GBF regulation and requirements for additional factors in this control process are not well understood.In an effort to identify potential GBF binding and control partners, maize GBF1 was used as bait in a yeast two-hybrid screen of an A. thaliana cDNA library. GBF Interacting Protein 1 (GIP1) arose from the screen as a 496 amino acid protein with a predicted molecular weight of 53,748 kDa that strongly interacts with GBFs. Northern analysis of A. thaliana tissue suggests a 1.8-1.9 kb GIP1 transcript, predominantly in roots. Immunolocalization studies indicate that GIP1 protein is mainly localized to the nucleus. In vitro electrophoretic mobility shift assays using an Adh G-box DNA probe and recombinant A. thaliana GBF3 or maize GBF1, showed that the presence of GIP1 resulted in a tenfold increase in GBF DNA binding activity without altering the migration, suggesting a transient association between GIP1 and GBF. Addition of GIP1 to intentionally aggregated GBF converted GBF to lower molecular weight macromolecular complexes and GIP1 also refolded denatured rhodanese in the absence of ATP. These data suggest GIP1 functions to enhance GBF DNA binding activity by acting as a potent nuclear chaperone or crowbar, and potentially regulates the multimeric state of GBFs, thereby contributing to bZIP-mediated gene regulation.

G-Box Binding Factor1 Reduces CATALASE2 Expression and Regulates the Onset of Leaf Senescence in Arabidopsis

“…Moreover, interaction with another protein, GBF-Interacting Protein1 (GIP1), resulted in a 10-fold increase in GBF1 DNA-binding activity without altering the migration in an electrophoretic mobility shift assay. This suggests a transient association of GIP1 and GBFs and a possible action as a potent nuclear chaperone or crowbar, potentially regulating the multimeric state of GBFs (Sehnke et al, 2005). Recently, it was shown that GBF1 is degraded by a proteasome-mediated pathway independent of COP1 and SPA1.…”

TF-Cluster: A pipeline for identifying functionally coordinated transcription factors via network decomposition of the shared coexpression connectivity matrix (SCCM)

“…Finally, Cluster 19 contains TFs that are involved in ABA signaling or response, an event incurred by water deprivation or salt stress. These TFs include GBF3 [84], ABF4 (Yoshida et al 2010), ANAC019 [85], ATHB7 [86], ATHB12 [86], ABF3 (Yoshida et al 2010), RD26[87], MYB102[88]. In this case, 47% of the TFs are associated with ABA signaling.…”

The Elucidation of the Interactome of 16 Arabidopsis bZIP Factors Reveals Three Independent Functional Networks

“…Recently, bZIP1 (S1 class) was described to follow a “hit-and-run” mode of action according to which it transiently binds to a large set of cis-elements [ 77 ], a model compatible with the dynamic exchange of monomers in the C/S1 network proposed here. Conversely, the function of the G and H networks dimers is broadly influenced by the cellular context, particularly via interactions with other regulatory factors [ 65 , 66 , 78 ]. In fact, the G-bZIPs feature a Proline Rich Domain, which is a protein-protein interaction domain and has been found to be required for their activity [ 11 , 66 , 79 , 80 ].…”