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Nuclear import of the parsley bZIP transcription factor CPRF2 is regulated by phytochrome photoreceptors.

Kircher S, Wellmer F, Nick P, Rügner A, Schäfer E, Harter K - J. Cell Biol. (1999)

Bottom Line: To understand these processes in light signal transduction we analyzed the three well-known members of the common plant regulatory factor (CPRF) family from parsley (Petroselinum crispum).Here, we demonstrate that these CPRFs, which belong to the basic- region leucine-zipper (bZIP) domain-containing transcription factors, are differentially distributed within parsley cells, indicating different regulatory functions within the regulatory networks of the plant cell.We suggest that light-induced nuclear import of CPRF2 is an essential step in phytochrome signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie II/Botanik, Universität Freiburg, 79104 Freiburg, Germany.

ABSTRACT
In plants, light perception by photoreceptors leads to differential expression of an enormous number of genes. An important step for differential gene expression is the regulation of transcription factor activities. To understand these processes in light signal transduction we analyzed the three well-known members of the common plant regulatory factor (CPRF) family from parsley (Petroselinum crispum). Here, we demonstrate that these CPRFs, which belong to the basic- region leucine-zipper (bZIP) domain-containing transcription factors, are differentially distributed within parsley cells, indicating different regulatory functions within the regulatory networks of the plant cell. In particular, we show by cell fractionation and immunolocalization approaches that CPRF2 is transported from the cytosol into the nucleus upon irradiation due to action of phytochrome photoreceptors. Two NH2-terminal domains responsible for cytoplasmic localization of CPRF2 in the dark were characterized by deletion analysis using a set of CPRF2-green fluorescent protein (GFP) gene fusion constructs transiently expressed in parsley protoplasts. We suggest that light-induced nuclear import of CPRF2 is an essential step in phytochrome signal transduction.

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(A) Schematic representation of functional domains  within CPRF2. P-rich, prolin-rich transactivation domain  (Rügner, A., and E. Schäfer, unpublished data). 1, retention domain 1 that has strong homology to the α-helical cytoplasmic retention domain (α-helix) of the mammalian heat shock factor 2.  2, acidic retention domain 2 that contains a CKII phosphorylation site as indicated (-SDDD-). NLS/bZIP, nuclear localization  sequence-containing basic-region leucine-zipper domain. N, NH2  terminus; C, COOH terminus; aa, amino acid. (B) Alternative  models of phy-regulated nuclear import of CPRF2 in parsley  cells. Pfr formation induces phosphorylation of the CKII site  within retention domain 2 of CPRF2. This modification either  abolishes intracellular masking of the NLS (model I) or results in  the release of CPRF2 from a cytoplasmic anchoring protein  (model II). In both cases, accession to the NLS results in nuclear  import, homo- and/or heterotypic CPRF2–protein interactions,  and binding of CPRF2-containing transcription factor complexes  to promoters of light-regulated genes. Further explanation in the  text. Pr, inactive form of phytochrome; Pfr, active form of phytochrome; ACGT, G-box like elements; x, y, z, cis-elements other  than G-box like motifs; see A for other abbreviations and notes.
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Figure 6: (A) Schematic representation of functional domains within CPRF2. P-rich, prolin-rich transactivation domain (Rügner, A., and E. Schäfer, unpublished data). 1, retention domain 1 that has strong homology to the α-helical cytoplasmic retention domain (α-helix) of the mammalian heat shock factor 2. 2, acidic retention domain 2 that contains a CKII phosphorylation site as indicated (-SDDD-). NLS/bZIP, nuclear localization sequence-containing basic-region leucine-zipper domain. N, NH2 terminus; C, COOH terminus; aa, amino acid. (B) Alternative models of phy-regulated nuclear import of CPRF2 in parsley cells. Pfr formation induces phosphorylation of the CKII site within retention domain 2 of CPRF2. This modification either abolishes intracellular masking of the NLS (model I) or results in the release of CPRF2 from a cytoplasmic anchoring protein (model II). In both cases, accession to the NLS results in nuclear import, homo- and/or heterotypic CPRF2–protein interactions, and binding of CPRF2-containing transcription factor complexes to promoters of light-regulated genes. Further explanation in the text. Pr, inactive form of phytochrome; Pfr, active form of phytochrome; ACGT, G-box like elements; x, y, z, cis-elements other than G-box like motifs; see A for other abbreviations and notes.

Mentions: The demonstration that the disappearance of CPRF2– GFP from the cytosol of parsley cells in response to light is due to active transport into the nucleus allowed us to perform a deletion analysis to map the retention domain(s). The removal of the entire NH2 terminus up to aa 159 abolished cytosolic retention in darkness whereas the aa80CPRF2–GFP fusion protein was still detectable in the cytosol. A similar loss of cytosolic localization was found when a short internal stretch between aa 178 and 192 (ΔCPRF2–GFP) was removed from the full-length protein. The deletion analysis suggests that CPRF2 contains two separable domains for cytoplasmic retention (see Fig. 6 A). In addition, the aa stretch containing both retention domains can be functionally transferred to heterologous bZIP factors (e.g., CPRF1) not found in the cytoplasmic compartment of evacuolated parsley protoplasts (Kircher, S., unpublished data). It is noteworthy that neither CPRF1 nor CPRF4 contain the sequence motifs that could be homologous to the retention domains of CPRF2.


Nuclear import of the parsley bZIP transcription factor CPRF2 is regulated by phytochrome photoreceptors.

Kircher S, Wellmer F, Nick P, Rügner A, Schäfer E, Harter K - J. Cell Biol. (1999)

(A) Schematic representation of functional domains  within CPRF2. P-rich, prolin-rich transactivation domain  (Rügner, A., and E. Schäfer, unpublished data). 1, retention domain 1 that has strong homology to the α-helical cytoplasmic retention domain (α-helix) of the mammalian heat shock factor 2.  2, acidic retention domain 2 that contains a CKII phosphorylation site as indicated (-SDDD-). NLS/bZIP, nuclear localization  sequence-containing basic-region leucine-zipper domain. N, NH2  terminus; C, COOH terminus; aa, amino acid. (B) Alternative  models of phy-regulated nuclear import of CPRF2 in parsley  cells. Pfr formation induces phosphorylation of the CKII site  within retention domain 2 of CPRF2. This modification either  abolishes intracellular masking of the NLS (model I) or results in  the release of CPRF2 from a cytoplasmic anchoring protein  (model II). In both cases, accession to the NLS results in nuclear  import, homo- and/or heterotypic CPRF2–protein interactions,  and binding of CPRF2-containing transcription factor complexes  to promoters of light-regulated genes. Further explanation in the  text. Pr, inactive form of phytochrome; Pfr, active form of phytochrome; ACGT, G-box like elements; x, y, z, cis-elements other  than G-box like motifs; see A for other abbreviations and notes.
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Figure 6: (A) Schematic representation of functional domains within CPRF2. P-rich, prolin-rich transactivation domain (Rügner, A., and E. Schäfer, unpublished data). 1, retention domain 1 that has strong homology to the α-helical cytoplasmic retention domain (α-helix) of the mammalian heat shock factor 2. 2, acidic retention domain 2 that contains a CKII phosphorylation site as indicated (-SDDD-). NLS/bZIP, nuclear localization sequence-containing basic-region leucine-zipper domain. N, NH2 terminus; C, COOH terminus; aa, amino acid. (B) Alternative models of phy-regulated nuclear import of CPRF2 in parsley cells. Pfr formation induces phosphorylation of the CKII site within retention domain 2 of CPRF2. This modification either abolishes intracellular masking of the NLS (model I) or results in the release of CPRF2 from a cytoplasmic anchoring protein (model II). In both cases, accession to the NLS results in nuclear import, homo- and/or heterotypic CPRF2–protein interactions, and binding of CPRF2-containing transcription factor complexes to promoters of light-regulated genes. Further explanation in the text. Pr, inactive form of phytochrome; Pfr, active form of phytochrome; ACGT, G-box like elements; x, y, z, cis-elements other than G-box like motifs; see A for other abbreviations and notes.
Mentions: The demonstration that the disappearance of CPRF2– GFP from the cytosol of parsley cells in response to light is due to active transport into the nucleus allowed us to perform a deletion analysis to map the retention domain(s). The removal of the entire NH2 terminus up to aa 159 abolished cytosolic retention in darkness whereas the aa80CPRF2–GFP fusion protein was still detectable in the cytosol. A similar loss of cytosolic localization was found when a short internal stretch between aa 178 and 192 (ΔCPRF2–GFP) was removed from the full-length protein. The deletion analysis suggests that CPRF2 contains two separable domains for cytoplasmic retention (see Fig. 6 A). In addition, the aa stretch containing both retention domains can be functionally transferred to heterologous bZIP factors (e.g., CPRF1) not found in the cytoplasmic compartment of evacuolated parsley protoplasts (Kircher, S., unpublished data). It is noteworthy that neither CPRF1 nor CPRF4 contain the sequence motifs that could be homologous to the retention domains of CPRF2.

Bottom Line: To understand these processes in light signal transduction we analyzed the three well-known members of the common plant regulatory factor (CPRF) family from parsley (Petroselinum crispum).Here, we demonstrate that these CPRFs, which belong to the basic- region leucine-zipper (bZIP) domain-containing transcription factors, are differentially distributed within parsley cells, indicating different regulatory functions within the regulatory networks of the plant cell.We suggest that light-induced nuclear import of CPRF2 is an essential step in phytochrome signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie II/Botanik, Universität Freiburg, 79104 Freiburg, Germany.

ABSTRACT
In plants, light perception by photoreceptors leads to differential expression of an enormous number of genes. An important step for differential gene expression is the regulation of transcription factor activities. To understand these processes in light signal transduction we analyzed the three well-known members of the common plant regulatory factor (CPRF) family from parsley (Petroselinum crispum). Here, we demonstrate that these CPRFs, which belong to the basic- region leucine-zipper (bZIP) domain-containing transcription factors, are differentially distributed within parsley cells, indicating different regulatory functions within the regulatory networks of the plant cell. In particular, we show by cell fractionation and immunolocalization approaches that CPRF2 is transported from the cytosol into the nucleus upon irradiation due to action of phytochrome photoreceptors. Two NH2-terminal domains responsible for cytoplasmic localization of CPRF2 in the dark were characterized by deletion analysis using a set of CPRF2-green fluorescent protein (GFP) gene fusion constructs transiently expressed in parsley protoplasts. We suggest that light-induced nuclear import of CPRF2 is an essential step in phytochrome signal transduction.

Show MeSH
Related in: MedlinePlus