YEAST TRANSCRIPTIONAL ACTIVATOR INO2 INTERACTS AS AN INO2P INO4P BASIC HELIX-LOOP-HELIX HETEROMERIC COMPLEX WITH THE INOSITOL CHOLINE-RESPONSIVE ELEMENT NECESSARY FOR EXPRESSION OF PHOSPHOLIPID BIOSYNTHETIC GENES IN SACCHAROMYCES-CEREVISIAE/

Coordinate transcriptional control of yeast genes involved in phosphol ipid biosynthesis is mediated by the inositol/choline-responsive eleme nt (ICRE) contained in the respective promoter regions. Regulatory gen es INO2 and INO4, both encoding basic helix-loop-helix (bHLH) proteins , are necessary for ICRE-dependent gene activation. By the use of size variants and by heterologous expression in E.coli we demonstrate that Ino2p and Ino4p are both necessary and sufficient for the formation o f the previously described FAS binding factor 1, Fbf1, interacting wit h the ICRE. Formation of a heteromeric complex between Ino2p and Ino4p by means of the respective bHLH domains was demonstrated in vivo by t he interaction of appropriate two-hybrid constructs and in vitro by Fa r-Western analyses. Neither Ino2p nor Ino4p binds to the ICRE as a hom odimer. When fused to the DNA-binding domain of GaI4p, Ino2p but not I no4p was able to activate a UAS(GAL)-containing reporter gene even in the absence of the heterologous Fbf1 subunit. By deletion studies, two separate transcriptional activation domains were identified in the N- terminal part of Ino2p. Thus, the bHLH domains of Ino2p and Ino4p cons titute the dimerization/DNA-binding module of Fbf1 mediating its inter action with the ICRE, while transcriptional activation is effected exc lusively by Ino2p.