REDOX SIGNALS IN MOLECULAR ADAPTATION

Avihai Danon, Tal Alergand , Yael Katz, Alexander Levitan, Eti Meir , Yaron Pereg, Tova Trebitsh.

Department of Plant Sciences, Tel. 972-8-934-2382, Fax. 972-8-934-4181, e-mail: Avihai.Danon@weizmann.ac.il

Objectives of Research:

Elucidation of redox-modulated signal transduction pathway regulating post-transcriptional gene expression - Translation of several chloroplast mRNAs requires the specific interaction of nuclear-encoded protein factors with RNA-elements located in the 5'-untranslated region of the chloroplast mRNAs. The RNA-binding activity of these regulatory proteins is light-regulated and has been shown to be under control of redox in vitro and in vivo. Our goal is to identify the factors involved in redox-transduction and to characterize their mode of function.

Recent Findings:

Light stimulates translation of chloroplast psbA mRNA within 5 minutes of illumination probably by activating a protein complex associated with the 5í untranslated region of this message. The protein complex contains a regulatory redox-active site responsive to thioredoxin. The regulatory redox-active site is characterized as two vicinal (neighbor) cysteines. We identified RB60, a protein disulfide isomerase-like member of the protein complex, as carrying the redox-active regulatory site, comprised of vicinal dithiol (VDS). From these experiments, we proposed that RB60 acts as sensor protein responsive to the ëlight signalí which is transduced as a reductive signal by thioredoxin. This suggests that for perception of the reductive signal in vivo, the regulatory VDS of RB60 has to be initially oxidized. We established, in parallel, the redox state in organello of the regulatory VDS and redox effects on D1 synthesis in intact chloroplasts. We have found that light activated specific oxidation of RB60 on one hand, and reduced RB60, probably via the ferredoxin-thioredoxin system, on the other. Higher light intensities increased the pool of reduced RB60 and the rate of psbA mRNA translation, suggesting that a counter-balanced action of reducing and oxidizing activities modulate translation of psbA mRNA in parallel with fluctuating light intensities. In the dark, chemical reduction of the vicinal dithiol site did not activate translation. This suggests a mechanism by which light primes redox-regulated translation by a yet unknown mechanism, and then the rate of translation is determined by the reduction-oxidation of a sensor protein (RB60) located in a complex bound to the 5í untranslated region of the chloroplastic mRNA (Figure).

VDS-containing proteins have been shown to participate in regulation of diverse and important biological activities, such as tyrosine kinases, receptors, enzymes of the reductive pentose phosphate cycle and translation. However it is yet unclear how the VDS-containing proteins mediate signal transduction and regulation by redox, how redox signals are transmitted in an otherwise redox-buffered milieu (under nonstress conditions), and how regulation specificity is attained. The relatively high abundance of the regulatory VDS-containing proteins in chloroplasts and fast kinetics of light signal transduction and activation of translation presents an excellent system for isolating and characterizing the function of these regulatory redox-active factors.

Recent Publications:

  1. Danon, A. and Mayfield, S.P. 1994. ADP-dependent phosphorylation regulates RNA-binding in vitro: Implications in light-modulated translation. EMBO J. 13:(9) 2227-2235.
  2. Mayfield, S.P., Cohen A, Danon, A. and Yohn, C. B. 1994. Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5í-untranslated region. J. Cell Biol. 127:1537-1545.
  3. Danon, A. and Mayfield, S.P. 1994. Light-regulated translation of chloroplast mRNAs through redox potential. Science 266:1717-1719.
  4. Yohn, C.B., Cohen, A., Danon, A. and Mayfield, S.P. 1996. Altered mRNA binding activity and decreased translation initiation in a nuclear mutant lacking translation of the chloroplast psbA mRNA. Mol. Cell. Biol. 16:3560-3566.
  5. Danon, A. 1997.Translational regulation in the Chloroplast. Plant Physiol. 115: 1293-1298.
  6. Yohn, C.B., Cohen, A., Danon, A., and Mayfield, S.P. 1988. A poly(A) binding protein functions in the chloroplast as a message specific translational factor. Proc. Natl. Acad. Sci. USA 95:2238-2243
  1. Trebitsh, T., Levitan, A., Sofer, A., and Danon, A. 1999. Translation of chloroplast psbA mRNA is modulated in the light by counter-acting oxidizing and reducing activities. Mol. Cell. Biol. In Press
Figure 1. A model depicting the redox transduction pathway controlling light-regulated translation. Binding of a protein complex acting as translational activator to the 5í untranslated region of psbA mRNA is required for its translation. Light regulates the binding of the regulatory protein complex via a prerequisite (priming) redox-independent pathway and a redox-dependent pathway. The priming pathway probably involves dephosphorylation and specific oxidation of RB60, rendering RB60 receptive to the reductive signal transduced by the redox-dependent pathway. In the redox-dependent pathway, light energy is captured by the thylakoids associated photosynthetic complexes to produce reducing potential. Two reducing equivalents are transferred in a series of oxidation-reduction reactions, involving ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin to reduce the regulatory vicinal dithiol site of RB60. Once reduced RB60 activates the binding of the protein complex to the 5í untranslated region of the message. Hence in the light, a counter-balanced action of reducing and oxidizing activities modulates the redox state of the pool of RB60, and consequently translation of psbA mRNA, in parallel with fluctuating light intensities.

Acknowledgements

A.D. holds The Judith and Martin Freedman Career Developmental Chair. This work was supported by a grant from Dorot Science Fellowships Foundation and grant from Minerva Foundation

Field of Research - Please indicate first and second priorities from the list below:

1) Plant sciences

2) Signal transduction