The Balance of Nuclear Import and Export Determines the Intracellular Distribution and Function of Tomato Heat Stress Transcription Factor HsfA2

Abstract

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...

In the complex family of heat stress transcription factors, HsfA1 has a unique role as master regulator of thermotolerance in tomato

“…In support of such a model, HsfA2 was characterized as a potent activator of reporter gene expression in general (Treuter et al 1993;Scharf et al 1998;Döring et al 2000;Heerklotz et al 2001). Moreover, in the test situation of protoplasts from CS plants, the defect of the hs response could be repaired by plasmid-borne HsfA2 expression.…”

“…Based on our results with the CS plants, we must conclude that the deficiency of HsfA1 expression cannot be complemented by any of the other closely related tomato Hsfs. Although our knowledge is still very limited, the functional analysis of the four tomato Hsfs cloned so far supports the concept of a marked diversification (Scharf et al 1990(Scharf et al , 1998Treuter et al 1993;Boscheinen et al 1997;Lyck et al 1997;Bharti et al 2000;Döring et al 2000;Heerklotz et al 2001). Evidently, each of these Hsfs has its own role in a complex network determined by the pattern of expression, intracellular localization, oligomerization behavior, activator function, and interaction of Hsfs with other proteins (for review, see Nover et al 2001).…”

“…6-8) was not anticipated. Usually, the strong nuclear export signal at the C terminus of HsfA2 precludes a significant retention in the nucleus unless it exists in heterooligomers with HsfA1 (Scharf et al 1998;Heerklotz et al 2001). We assume that other class A Hsfs of tomato may fulfill this function.…”

“…(Pelham 1982;Pelham and Bienz 1982;Nover 1987). Similar to other transcription factors, Hsfs have a modular structure with an N-terminal DNA-binding domain characterized by a central helix-turn-helix motif, an adjacent oligomerization domain with a bipartite heptad pattern of hydrophobic amino acid residues (HR-A/B region), and sequence motifs essential for nuclear import and export (NLS, NES;Wu 1995;Morimoto 1998;Heerklotz et al 2001;Nover et al 2001). In many cases, the C-terminal activation domains are characterized by short peptide motifs (AHA motifs) shown to be crucial for the activator function (Döring et al 2000).…”

Analysis of the nuclear localization signal of TRF1 in non-small cell lung cancer

“…Nucleocytoplasmic redistribution of proteins involved in NLS and NES is evidently an important process in many systems [Heerklotz et al, 2001]. NES is a short amino acid sequence of 5-6 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex.…”