ABSTRACT
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.
Subject(s)
DNA-Binding Proteins/metabolism , Heat-Shock Proteins/metabolism , Plant Proteins/metabolism , Solanum lycopersicum/metabolism , Transcription Factors/metabolism , Arabidopsis Proteins , Base Sequence , Cytoplasm/metabolism , DNA Primers , Electrophoresis, Polyacrylamide Gel , Heat Shock Transcription Factors , Heat-Shock Proteins/genetics , Heat-Shock Proteins/isolation & purification , Hot Temperature , Solanum lycopersicum/genetics , Molecular Sequence Data , Plant Proteins/genetics , Plant Proteins/isolation & purification , Protoplasts/metabolism , Nicotiana/metabolismABSTRACT
Biosynthesis of the tetrahydrobiopterin (BH(4)) cofactor, essential for catecholamines and serotonin production and nitric oxide synthase (NOS) activity, requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Upon studying the distribution of GTPCH and PTPS with polyclonal immune sera in cross sections of rat brain, prominent nuclear staining in many neurons was observed besides strong staining in peri-ventricular structures. Furthermore, localization studies in transgenic mice expressing a Pts-LacZ gene fusion containing the N-terminal 35 amino acids of PTPS revealed beta-galactosidase in the nucleus of neurons. In contrast, PTPS-beta-galactosidase was exclusively cytoplasmic in the convoluted kidney tubules but nuclear in other parts of the nephron, indicating again that nuclear targeting may occur only in specific cell categories. Furthermore, the N terminus of PTPS acts as a domain able to target the PTPS-beta-galactosidase fusion protein to the nucleus. In transiently transfected COS-1 cells, which do not express GTPCH and PTPS endogenously, we found cytoplasmic and nuclear staining for GTPCH and PTPS. To further investigate nuclear localization of all three BH(4)-biosynthetic enzymes, we expressed Flag-fusion proteins in transiently transfected COS-1 cells and analyzed the distribution by immunolocalization and sub-cellular fractionation using anti-Flag antibodies and enzymatic assays. Whereas 5-10% of total GTPCH and PTPS and approximately 1% of total SR were present in the nucleus, only GTPCH was confirmed to be an active enzyme in nuclear fractions. The in vitro studies together with the tissue staining corroborate specific nuclear localization of BH(4)-biosynthetic proteins with yet unknown biological function.