Heat stress transcription factors from tomato can functionally replace HSF1 in the yeast Saccharomyces cerevisiae.

The fact that yeast HSF1 is essential for survival under nonstress conditions can be used to test heterologous Hsfs for the ability to substitute for the endogenous protein. Our results demonstrate that like Hsf of Drosophila, tomato Hsfs A1 and A2 can functionally replace the corresponding yeast protein, but Hsf B1 cannot. In addition to survival at 28 degrees C, we checked the transformed yeast strains for temperature sensitivity of growth, induced thermotolerance and activator function using two different lacZ reporter constructs. Tests with full-length Hsfs were supplemented by assays using mutant Hsfs lacking parts of their C-terminal activator region or oligomerization domain, or containing amino acid substitutions in the DNA-binding domain. Remarkably, results with the yeast system are basically similar to those obtained by the analysis of the same Hsfs as transcriptional activators in a tobacco protoplast assay. Most surprising is the failure of HsfB1 to substitute for the yeast Hsf. The defect can be overcome by addition to HsfB1 of a short C-terminal peptide motif from HsfA2 (34 amino acid residues), which represents a type of minimal activator necessary for interaction with the yeast transcription apparatus. Deletion of the oligomerization domain (HR-A/B) does not interfere with Hsf function for survival or growth at higher temperatures. But monomeric Hsf has a markedly reduced affinity for DNA, as shown by lacZ reporter and band-shift assays.

Functional immobilization of a DNA-binding protein at a membrane interface via histidine tag and synthetic chelator lipids.

The coupling of a DNA-binding protein to self-organized lipid monolayers is examined at the air-water interface by means of film balance techniques and epifluorescence microscopy. We used two recombinant species of the heat shock factor HSF24 which differ only in a carboxy-terminal histidine tag that interacts specifically with the nickel-chelating head group of a synthetic chelator lipid. As key function, HSF24 binds to DNA that contains heat-shock responsible promoter elements. In solution, DNA-protein complex formation is demonstrated for the wild type and fusion protein. Substantial questions of these studies are whether protein function is affected after adsorption to lipid layers and whether a specific docking via histidine tag to the chelator lipid leads to functional immobilization. Using lipid mixtures that allow a lateral organization of chelator lipids within the lipid film, specific binding and unspecific adsorption can be distinguished by pattern formation of DNA-protein complexes. At the lipid interface, functional DNA-protein complexes are only detected, when the histidine-tagged protein was immobilized specifically to a chelator lipid containing monolayer. These results demonstrate that the immobilization of histidine-tagged biomolecules to membranes via chelator lipids is a promising approach to achieve a highly defined deposition of these molecules at an interface maintaining their function.