The balance of nuclear import and export determines the intracellular distribution and function of tomato heat stress transcription factor HsfA2.
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 degrees C). Despite the fact that nuclear import and export are not markedly affected, HsfA2 remains completely cytoplasmic at 41 degrees C even in the presence of LMB. The temperature-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.
Department of Molecular Cell Biology, Biocenter, Goethe-University Frankfurt, Frankfurt am Main, Germany.
D
D
Heerklotz
P
P
Döring
F
F
Bonzelius
S
S
Winkelhaus
L
L
Nover
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D015153:Blotting, Western; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D008156:Luciferases / Q000378:metabolism; D018551:Solanum lycopersicum / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D010940:Plant Proteins; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013696:Temperature; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection; D014170:Transformation, Genetic
D000903:Antibiotics, Antineoplastic; D004268:DNA-Binding Proteins; D005231:Fatty Acids, Unsaturated; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D010940:Plant Proteins; D014157:Transcription Factors; D008156:Luciferases; C038753:leptomycin B