Figure 5
Expression and activity of tagged GSV transcription factors and dendrogram from protein–metabolite immunoisolation. (A) GSV translational fusion to a triple tag (6x-HIS (white), HA-epitope (dark blue), Protease 3C cleavage site (orange), and IgG binding domain ZZ (yellow)) in pBG1805 [33]. (B) Immunoisolation of tagged proteins with IgG Sepharose beads followed by SDS-PAGE. One of four biological replicates is shown. Y258 URA3+ (lane 1) served as the negative control, while GSV proteins are indicated by red asterisks. The GV fusion lacking the START domain is marked by a black asterisk. Arrowheads indicate IgG heavy and light chains. (C) Tagged GSV constructs were expressed in erg6 cells containing the LacZ reporter and transcriptional activity was measured in β-Gal units. Double asterisks indicate a significant increase in activity over the GV control (two-tailed t-test, P < 0.03). Error bars indicate standard deviations for two independent transformants in four trials. (D) Dendrogram from protein–metabolite immunoisolation. Two-way hierarchical clustering was performed on the metabolite enrichment data relative to the GV control with values expressed as the logarithm base 2 (ratio) of normalized metabolite intensities. Horizontal clusters represent START domain clusters based on protein-bound metabolite enrichment. Vertical clusters show groups of protein-bound metabolites clustered by START domain. Metabolite names marked by asterisks were validated by mass spectrometry, matching exact mass and retention time to a known standard. CE Metabolites of the same chemical subclass were grouped and given a superscript number to distinguish members that bear distinct masses and retention times. aa, amino acids; AD, activation domain; DBD, DNA binding domain; GL2, Glabra2; GSV, Gal4 DNA binding domain:START domain:VP16 activation domain; GV, GAL4 DNA binding domain:VP16 activation domain; IgG, immunoglobulin G; PCTP, phosphatidylcholine transfer protein; PDF2, Protodermal Factor 2; StAR, steroidogenic acute regulatory protein; START, StAR-related lipid transfer.