Dominant negative suppression of arabidopsis photoresponses by mutant phytochrome A sequences identifies spatially discrete regulatory domains in the photoreceptor.

We used the exaggerated short hypocotyl phenotype induced by oat phytochrome A overexpression in transgenic Arabidopsis to monitor the biological activity of mutant phytochrome A derivatives. Three different mutations, which were generated by removing 52 amino acids from the N terminus (delta N52), the entire C-terminal domain (delta C617), or amino acids 617-686 (delta 617-686) of the oat molecule, each caused striking dominant negative interference with the ability of endogenous Arabidopsis phytochrome A to inhibit hypocotyl growth in continuous far-red light ("far-red high irradiance response" conditions). By contrast, in continuous white or red light, delta N52 was as active as the unmutagenized oat phytochrome A protein in suppressing hypocotyl elongation, while delta C617 and delta 617-686 continued to exhibit dominant negative behavior under these conditions. These data suggest that at least three spatially discrete molecular domains coordinate the photoregulatory activities of phytochrome A in Arabidopsis seedlings. The first is the chromophore-bearing N-terminal domain between residues 53 and 616 that is apparently sufficient for the light-induced initiation but not the completion of productive interactions with transduction chain components. The second is the C-terminal domain between residues 617 and 1129 that is apparently necessary for completion of productive interactions under all irradiation conditions. The third is the N-terminal 52 amino acids that are apparently necessary for completion of productive interactions only under far-red high irradiance conditions and are completely dispensable under white and red light regimes.     

Beta-amyloid protein converting enzyme 1 and brain-specific type II membrane protein BRI3: binding partners processed by furin

Using a yeast two-hybrid system, we screened a human brain cDNA library for possible interacting proteins with the C-terminal cytosolic tail of the beta-secretase beta-amyloid protein converting enzyme (BACE)1. This identified seven potential candidates, including the brain-specific type II membrane protein BRI3. Co-localization and co-immunoprecipitation experiments confirmed that BACE1 and BRI3 co-localize and interact with each other via the cytosolic tail of BACE1. Furthermore, pulse and pulse-chase analyses revealed that the pro-protein convertases furin, and to a lesser extent PC7 and PC5A, process BRI3 into a C-terminal secreted approximately 4-kDa product. Thus, furin efficiently processes both pro-BACE1 and its novel interacting protein pro-BRI3.     

Over-expression of a C-terminal region of phytochrome B

In an attempt to identify domains directly involved in the signal transduction of phytochrome B (phyB), we over-expressed the achromophoric C-terminal half of phyB under control of the CaMV-35S promoter in transgenicArabidopsis. In three independent transgenic lines, we detected accumulation of the introduced protein of predicted size at levels higher than that of the endogenous phyB by immunoblot analysis. Although these transgenic plants did not show any phenotype in the dark, enhancement of the phyB-dependent inhibition of hypocotyl elongation and reduction of the phytochrome A (phyA)-dependent inhibition were observed.     

Specific interaction of the 70-kDa heat shock cognate protein with the tetratricopeptide repeats

Using a yeast two-hybrid system with the 70-kDa heat shock cognate protein (hsc70) or its C-terminal 30-kDa domain as baits, we isolated several proteins interacting with hsc70, including Hip/p48 and p60/Hop. Both are known to interact with hsc70. Except for Hip/p48, all of the proteins that we isolated interact with the 30-kDa domain. Moreover, the EEVD motif at the C terminus of the 30-kDa domain appears essential for this interaction. Sequence analysis of these hsc70-interacting proteins reveals that they all contain tetratricopeptide repeats. Using deletion mutants of these proteins, we demonstrated either by two-hybrid or in vitro binding assays that the tetratricopeptide repeat domains in these proteins are necessary and sufficient for mediating the interaction with hsc70.     

Tomato ringspot virus proteins containing the nucleoside triphosphate binding domain are transmembrane proteins that associate with the endoplasmic reticulum and cofractionate with replication complexes

Replication of all known positive-strand RNA viruses occurs in replication complexes associated with intracellular membranes. The putative nucleoside triphosphate binding (NTB) protein of Tomato ringspot virus (ToRSV) contains a stretch of hydrophobic residues at its C terminus, suggesting that it may act as a membrane anchor for the replication complex. Anti-NTB antibodies detected two predominant proteins in membrane-enriched fractions (the 66-kDa NTB and 69-kDa NTB-VPg proteins) along with other, larger proteins. The proteins containing the NTB domain cofractionated with markers of the endoplasmic reticulum (ER) and with ToRSV-specific RNA-dependent RNA polymerase activity in sucrose gradients. ToRSV infection induced severe changes in the morphology of the ER in plants expressing an ER-targeted green fluorescent protein (ER-GFP), and proteins containing the NTB domain colocalized with ER-GFP in indirect immunofluorescence assays. The proteins containing the NTB domain have properties of integral membrane proteins. Proteinase K protection assays using purified membranes from infected plants revealed that although the central portion of the NTB domain is exposed to the cytoplasmic face of the membranes, an 8-kDa fragment, recognized by anti-VPg antibodies, is protected by the membranes. This fragment probably consists of the 3-kDa VPg and the 5-kDa stretch of hydrophobic residues at the C terminus of the NTB protein, suggesting a luminal location for the VPg in at least a portion of the molecules. These results provide evidence that proteins containing the NTB domain are transmembrane proteins associated with ER-derived membranes and support the hypothesis that one or several of the proteins containing the NTB domain anchor the replication complex to the ER.     

Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein

Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.     

The RanBP2 zinc finger domains of chloroplast RNA editing factor OZ1 are required for protein–protein interactions and conversion of C to U

In the chloroplast, organelle zinc finger 1 (OZ1) is a RanBP2-type zinc finger (Znf) protein required for many RNA editing events, a process by which specific cytosines are enzymatically converted to uracils as a correction mechanism for missense mutations in the organelle genomes. RNA editing is carried out by a large multi-protein complex called the ‘editosome’ that contains members of the pentatricopeptide repeat (PPR) protein family, the RNA editing factor interacting protein (also known as MORF) family and the organelle RNA-recognition motif (ORRM) family, in addition to OZ1. OZ1 is an 82-kDa protein with distinct domains, including a pair of Znf domains and a unique C-terminal region. To elucidate the functions of these domains, we have generated truncations of OZ1 for use in protein–protein interaction assays that identified the C-terminal region of OZ1, as well as the Znf domains as the primary interactors with PPR proteins, which are factors required for site-specificity and enzymatic editing. Expression of these OZ1 truncations in vivo showed that the Znf domains were required to restore chloroplast RNA editing in oz1 knockout plants. Mutation of key structural residues in the Znf domains showed that they are necessary for editing and required for interaction with ORRM1, a general editing factor with an RNA-binding domain. These functional characterizations of the Znfs and novel C-terminal domain contribute to our understanding of the model for the chloroplast plant editosome.     

Identification of conserved residues required for the binding of a tetratricopeptide repeat domain to heat shock protein 90.

The sequential binding of heat shock protein 90 (hsp90) to a series of tetratricopeptide repeat (TPR) proteins is critical to its function as a molecular chaperone. We have used site-directed mutagenesis to clarify the structural basis for the binding of hsp90 to the TPR domain of phosphoprotein phosphatase 5 (PP5). This TPR domain was chosen for study because its three-dimensional structure is known. We examined co-immunoprecipitation of hsp90 with wild type and mutant TPR constructs from transfected cells. Only mutations located on one face of the TPR domain affected hsp90 binding. This allowed the identification of a binding groove. Three basic residues that are highly conserved in hsp90-binding TPR proteins extend prominently into this groove. Lys-97 and Arg-101 were absolutely required for hsp90 binding, while mutation of Arg-74 diminished, but did not abrogate, hsp90 binding. Mutation of Lys-32, another conserved basic residue in the binding groove, also blocked hsp90 binding. The TPR domain of PP5 bound specifically to a 12-kDa C-terminal fragment of hsp90. This binding was reduced by mutation of acidic residues in the hsp90 fragment. These data suggest conservation, among hsp90-binding TPR proteins, of a binding groove containing basic residues that interact with acidic residues near the C terminus of hsp90.     

Chimeric proteins between cry1 and cry2 Arabidopsis blue light photoreceptors indicate overlapping functions and varying protein stability.

A blue light (cryptochrome) photoreceptor from Arabidopsis, cry1, has been identified recently and shown to mediate a number of blue light-dependent phenotypes. Similar to phytochrome, the cryptochrome photoreceptors are encoded by a gene family of homologous members with considerable amino acid sequence similarity within the N-terminal chromophore binding domain. The two members of the Arabidopsis cryptochrome gene family (CRY1 and CRY2) overlap in function, but their proteins differ in stability: cry2 is rapidly degraded under light fluences (green, blue, and UV) that activate the photoreceptor, but cry1 is not. Here, we demonstrate by overexpression in transgenic plants of cry1 and cry2 fusion constructs that their domains are functionally interchangeable. Hybrid receptor proteins mediate functions similar to cry1 and include inhibition of hypocotyl elongation and blue light-dependent anthocyanin accumulation; differences in activity appear to be correlated with differing protein stability. Because cry2 accumulates to high levels under low-light intensities, it may have greater significance in wild-type plants under conditions when light is limited.     

Specificity of RCN1-mediated protein phosphatase 2A regulation in meristem organization and stress response in roots

Protein dephosphorylation by the serine/threonine protein phosphatase 2A (PP2A) modulates a broad array of cellular functions. PP2A normally acts as a heterotrimeric holoenzyme complex comprising a catalytic subunit bound by regulatory A and B subunits. Characterization of the regulatory A subunit isoforms (ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 [RCN1], PP2AA2, and PP2AA3) of Arabidopsis thaliana PP2A has shown that RCN1 plays a primary role in controlling root and hypocotyl PP2A activity in seedlings. Here we show that hypocotyl and root growth exhibit different requirements for RCN1-mediated regulation of PP2A activity. Roots of rcn1 mutant seedlings exhibit characteristic abnormalities in cell division patterns at the root apical meristem, as well as reduced growth under ionic, osmotic, and oxidative stress conditions. We constructed chimeric A subunit genes and found that restoration of normal root tip development in rcn1 plants requires both regulatory and coding sequences of RCN1, whereas the hypocotyl elongation defect of rcn1 plants can be complemented by either RCN1 or PP2AA3 transgenes. Furthermore, the RCN1 and PP2AA3 proteins exhibit ubiquitous subcellular localization patterns in seedlings and both associate with membrane compartments. Together, these results show that RCN1-containing PP2A has unique functions that cannot be attributed to isoform-specific expression and localization patterns. Postembryonic RCN1 function is required to maintain normal auxin distribution and stem cell function at the root apex. Our data show that RCN1-regulated phosphatase activity plays a unique role in regulating postembryonic root development and stress response.     

New insights in protein phosphorylation: a signature for protein phosphatase 1 interacting proteins

Protein phosphatase 1 is regulated by the interaction between a catalytic subunit (PP1c) and multiple interacting proteins that allow the specific dephosphorylation of diverse cellular targets. This communication proposes to use the simultaneous presence of distinct consensus PP1c docking motifs R/K-x(0,1)-V-x-F and F-x-x-R/K-x-R/K as a signature to identify proteins putatively interacting with the PP1c. To develop this concept, we propose a new website, http://pp1 signature.pasteur.fr, which allows the identification of putative PP1-interacting proteins containing the two distinct PP1c docking consensus motifs represented in the Swissprot library. To validate the new concept of signature, we were able to characterise, by co-immunoprecipitation, four new PP1c interacting proteins randomly selected from the database in our website.     

A novel high-throughput in vivo molecular screen for shade avoidance mutants identifies a novel phyA mutation

The shade avoidance syndrome (SAS) allows plants to anticipate and avoid shading by neighbouring plants by initiating an elongation growth response. The phytochrome photoreceptors are able to detect a reduction in the red:far red ratio in incident light, the result of selective absorption of red and blue wavelengths by proximal vegetation. A shade-responsive luciferase reporter line (PHYB::LUC) was used to carry out a high-throughput screen to identify novel SAS mutants. The dracula 1 (dra1) mutant, that showed no avoidance of shade for the PHYB::LUC response, was the result of a mutation in the PHYA gene. Like previously characterized phyA mutants, dra1 showed a long hypocotyl in far red light and an enhanced hypocotyl elongation response to shade. However, dra1 additionally showed a long hypocotyl in red light. Since phyB levels are relatively unaffected in dra1, this gain-of-function red light phenotype strongly suggests a disruption of phyB signalling. The dra1 mutation, G773E within the phyA PAS2 domain, occurs at a residue absolutely conserved among phyA sequences. The equivalent residue in phyB is absolutely conserved as a threonine. PAS domains are structurally conserved domains involved in molecular interaction. Structural modelling of the dra1 mutation within the phyA PAS2 domain shows some similarity with the structure of the phyB PAS2 domain, suggesting that the interference with phyB signalling may be the result of non-functional mimicry. Hence, it was hypothesized that this PAS2 residue forms a key distinction between the phyA and phyB phytochrome species.     

Interaction of the protein phosphatase 2A with the regulatory domain of the cystic fibrosis transmembrane conductance regulator channel

A direct interaction of the regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator protein (CFTR) with PR65, a regulatory subunit of the protein phosphatase 2A (PP2A), was shown in yeast two hybrid, pull-down and co-immunoprecipitation experiments. The R domain could be dephosphorylated by PP2A in vitro. Overexpression of the interacting domain of PR65 in Caco-2 cells, as well as treatment with okadaic acid, showed a prolonged deactivation of the chloride channel. Taken together our results show a direct and functional interaction between CFTR and PP2A.