Involvement of Arabidopsis ROF2 (FKBP65) in thermotolerance.

The ROF2 (FKBP65) is a heat stress protein which belongs to the FK506 Binding Protein (FKBP) family. It is homologous to ROF1 (FKBP62) which was recently shown to be involved in long term acquired thermotolerance by its interaction with HSP90.1 and modulation of the heat shock transcription factor HsfA2. In this study, we have demonstrated that ROF2 participates in long term acquired thermolerance, its mode of action being different from ROF1. In the absence of ROF2, the small heat shock proteins were highly expressed and the plants were resistant to heat stress, opposite to the effect observed in the absence of ROF1. It was further demonstrated that ROF2 transcription is modulated by HsfA2 which is also essential for keeping high levels of ROF2 during recovery from heat stress. ROF2 localization to the nucleus was observed several hours after heat stress exposure and its translocation to the nucleus was independent from the presence of HSP90.1 or HsfA2. ROF2 has been shown to interact with ROF1, to form heterodimers and it is suggested that via this interaction it can join the complex ROF1-HSP90.1- HsfA2. Transient expression of ROF2 together with ROF1 repressed transcription of small HSPs. A model describing the mode of action of ROF2 as a heat stress modulator which functions in negative feedback regulation of HsfA2 is proposed.

Arabidopsis immunophilins ROF1 (AtFKBP62) and ROF2 (AtFKBP65) exhibit tissue specificity, are heat-stress induced, and bind HSP90.

The plant co-chaperones FK506-binding proteins (FKBPs) are peptidyl prolyl cis-trans isomerases that function in protein folding, signal transduction and chaperone activity. We report the characterization of the Arabidopsis large FKBPs ROF1 (AtFKBP62) and ROF2 (AtFKBP65) expression and protein accumulation patterns. Transgenic plants expressing ROF1 promoter fused to GUS reporter gene reveal that ROF1 expression is organ specific. High expression was observed in the vascular elements of roots, in hydathodes and trichomes of leaves and in stigma, sepals, and anthers. The tissue specificity and temporal expression of ROF1 and ROF2 show that they are developmentally regulated. Although ROF1 and ROF2 share 85% identity, their expression in response to heat stress is differentially regulated. Both genes are induced in plants exposed to 37 degrees C, but only ROF2 is a bonafide heat-stress protein, undetected when plants are grown at 22 degrees C. ROF1/ROF2 proteins accumulate at 37 degrees C, remain stable for at least 4 h upon recovery at 22 degrees C, whereas, their mRNA level is reduced after 1 h at 22 degrees C. By protein interaction assays, it was demonstrated, that ROF1 is a novel partner of HSP90. The five amino acids identified as essential for recognition and interaction between the mammalian chaperones and HSP90 are conserved in the plant ROF1-HSP90. We suggest that ROF/HSP90 complexes assemble in vivo. We propose that specific complexes formation between an HSP90 and ROF isoforms depends on their spatial and temporal expression. Such complexes might be regulated by environmental conditions such as heat stress or internal cues such as different hormones.

Biochemical support for the V-ATPase rotary mechanism: antibody against HA-tagged Vma7p or Vma16p but not Vma10p inhibits activity.

V-ATPase null mutants in yeast have a distinct, conditionally lethal phenotype that can be obtained through disruption of any one of its subunits. This enables supplementation of this mutant with the relevant subunit tagged with an epitope against which an antibody is available. In this system, the effect of antibody on the activity of the enzyme can be analyzed. Towards this end we used HA to tag subunits Vma7p, Vma10p and Vma16p, which are assumed to represent, respectively, the shaft, stator and turbine of the enzyme, and used them to supplement the corresponding yeast V-ATPase null mutants. The anti-HA epitope antibody inhibited both the ATP-dependent proton uptake and the ATPase activities of the Vma16p-HA and Vma7p-HA containing complexes, in intact vacuoles and in the detergent-solubilized enzyme. Neither of these activities was inhibited by the antibody in Vma10p-HA containing enzyme. These results support the function of Vma10p as part of the stator, while the other tagged subunits are part of the rotor apparatus. The HA-tag was attached to the N terminus of Vma16p; thus the antibody inhibition points to its accessibility outside the vacuolar membrane. This assumption is supported by the supplementation of the yeast mutant by the homologues of Vma16p isolated from Arabidopsis thaliana and lemon fruit c-DNA. Contrary to yeast, which has five predicted helices, the plant subunit Vma16p has only four. Our results confirm a recent report that only four of the yeast Vma16p complexes are actually transmembrane helices.