Tomato heat stress transcription factor HsfA2 is a shuttling protein with dominant cytoplasmic localization as a result of a nuclear import combined with an efficient export. Besides the nuclear localization signal (NLS) adjacent to the oligomerization domain, a C-terminal leucine-rich motif functions as a nuclear export signal (NES). Mutant forms of HsfA2 with a defective or an absent NES are nuclear proteins. The same is true for the wild-type HsfA2 if coexpressed with HsfA1 or in the presence of export inhibitor leptomycin B (LMB). Fusion of the NES domain of HsfA2 to HsfB1, which is a nuclear protein, caused export of the HsfB1-A2NES hybrid protein, and this effect was reversed by the addition of LMB. Due to the lack of background problems, Chinese hamster ovary (CHO) cells represent an excellent system for expression and functional analysis of tomato Hsfs. The results faithfully reflect the situation found in plant cells (tobacco protoplasts). The intriguing role of NLS and NES accessibility for the intracellular distribution of HsfA2 is underlined by the results of heat stress treatments of CHO cells (41°C). Despite the fact that nuclear import and export are not markedly affected, HsfA2 remains completely cytoplasmic at 41°C even in the presence of LMB. The temper- ature-dependent conformational transition of HsfA2 with shielding of the NLS evidently needs intramolecular interaction between the internal HR-A/B and the C-terminal HR-C regions. It is not observed with the HR oligomerization domain (HR-A/B region) deletion form of HsfA2 or in HsfA2-HsfA1 hetero-oligomers.Key regulators of the heat stress (HS) response are the HS transcription factors (Hsfs), which belong to a family of proteins conserved throughout the eukaryotic kingdom (24,26,35,46). Hsfs have a modular structure with an N-terminal DNAbinding domain characterized by a helix-turn-helix motif, an adjacent domain with heptad hydrophobic repeats (HR-A/B) involved in oligomerization, a cluster of basic amino acid residues essential for nuclear import (the nuclear localization signal, or NLS) and a C-terminal activation domain (Fig. 1).The high degree of structural and functional conservation of Hsfs was documented repeatedly by using heterologous systems for Hsf expression in combination with appropriate reporter assays. Thus, Drosophila melanogaster and human Hsfs were tested in plant cells, Xenopus oocytes, and Saccharomyces cerevisiae (5,6,22,43,50), and plant Hsfs were tested in yeast, Drosophila, and human cells (4,5,8,17). Using yeast strains with disruption of the endogenous Hsf1 gene, it was shown that many of these heterologous Hsfs were able to replace the yeast Hsf1 in most of its functions, i.e., in Hsf-dependent reporter assays, in the survival function both at 25 and 37°C, and in the generation of the thermotolerant state (5,12,22,48).In plants, the Hsf system is more complex than in any other organisms investigated so far (26,28,35). (i) Besides the constitutively expressed members of the Hsf family, many Hsfs themselves are HS-i...
HsfA2 is a heat stress (hs)-induced Hsf in peruvian tomato (Lycopersicon peruvianum) and the cultivated form Lycopersicon esculentum. Due to the high activator potential and the continued accumulation during repeated cycles of heat stress and recovery, HsfA2 becomes a dominant Hsf in thermotolerant cells. The formation of heterooligomeric complexes with HsfA1 leads to nuclear retention and enhanced transcriptional activity of HsfA2. This effect seems to represent one part of potential molecular mechanisms involved in its activity control. As shown in this paper, the activity of HsfA2 is also controlled by a network of nucleocytoplasmic small Hsps influencing its solubility, intracellular localization and activator function. By yeast two-hybrid interaction and transient coexpression studies in tobacco (Nicotiana plumbaginifolia) mesophyll protoplasts, we found that tomato (Lycopersicon esculentum) Hsp17.4-CII acts as corepressor of HsfA2. Given appropriate conditions, both proteins together formed large cytosolic aggregates which could be solubilized in presence of class CI sHsps. However, independent of the formation of aggregates or of the nucleocytoplasmic distribution of HsfA2, its transcriptional activity was specifically repressed by interaction of Hsp17.4-CII with the C-terminal activator domain. Although not identical in all aspects, the situation with the highly expressed, heat stress-inducible Arabidopsis HsfA2 was found to be principally similar. In corresponding reporter assays its activity was repressed in presence of AtHsp17.7-CII but not of AtHsp17.6-CII or LpHsp17.4-CII.
SummaryStress-induced transcription of heat shock proteins (Hsps) in eukaryotes is mediated by a conserved class of transcription factors called heat stress transcription factors (Hsfs). Here we report the isolation and functional characterization of HsfA3, a new member of the Hsf family. HsfA3 was cloned from a tomato heat stress cDNA library by yeast two-hybrid screening, using HsfA1 as a bait. HsfA3 is a singlecopy gene with all the conserved sequence elements characteristic of a heat stress transcription factor. The constitutively expressed HsfA3 is mainly found in the cytoplasm under control conditions and in the nucleus under heat stress conditions. Functionally, HsfA3 behaves similarly to the already known members of tomato Hsf family. It is able to substitute yeast Hsf for viability functions and is a strong activator of Hsf-dependent reporter constructs both in tobacco protoplasts and yeast. Finally, similar to the AHA motifs in HsfA1 and HsfA2, the activator function depends on four short peptide motifs with a central tryptophan residue found in the C-terminal domain of HsfA3.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.