Nuclear localization of the plant protein Ser/Thr phosphatase PP7.

Recently we identified novel plant Ser/Thr phosphatases, termed PP7, which belong to the PPP family and have no known close homologs in other kingdoms. We now addressed the intracellular location of Arabidopsis thaliana PP7 using GFP fusions and confocal laser scanning microscopy. PP7. GFP fusion was expressed transiently or stably in Nicotiana benthamiana. PP7. GFP was found to be a predominantly nuclear protein. Effects of cytoskeleton-disrupting drugs indicate that cytoskeleton may be required for efficient PP7. GFP delivery to the nucleus. Deletion of a potential nuclear localization signal in the first insert in the catalytic domain, as well as exposure to the dark, cold, high salinity and abscisic acid failed to prevent nuclear localization of PP7. GFP. Deletion of the 44 C-terminal amino acids resulted in a fusion protein located exclusively in the cytoplasm. The results suggest a possible similarity of the nuclear targeting signals in PP7 and the PP5/PPT subfamily.

Purification of plant protein phosphatase PP7 and evidence for its redox regulation.

PP7, a recently identified protein Ser/Thr phosphatase of the PPP family distantly related to phosphatases PP5/PPT and PPEF/rdgC, was purified from cauliflower extracts to apparent homogeneity. Purified cauliflower PP7 and recombinant PP7 expressed in Escherichia coli exhibit light absorption in the visible range with a maximum at approximately 430 nm. Under nonreducing conditions, native PP7 exists as a mixture of monomer with an intramolecular disulfide bridge, disulfide-linked homodimer, and possibly disulfide-linked complexes with potential partner proteins. The activity of recombinant Arabidopsis thaliana PP7 is reversibly regulated by redox agents. The results demonstrate the existence of PP7 protein in planta and suggest a possibility of redox regulation of this protein phosphatase.

Interaction of plant protein Ser/Thr phosphatase PP7 with calmodulin.

We have recently identified PP7, a novel group of plant protein Ser/Thr phosphatases, and hypothesized that PP7 may possess a calmodulin-binding site. To test this hypothesis, we assessed the effect of calmodulin on the activity of recombinant Arabidopsis thaliana PP7 and directly tested interaction between PP7 and calmodulin using surface plasmon resonance. Calmodulin exerted a moderate inhibitory effect on the phosphatase activity of PP7 with submicromolar affinity. PP7 specifically interacted with immobilized calmodulin (but not with recoverin, another EF hand Ca(2+)-binding protein) in a strictly Ca(2+)-dependent manner with nanomolar affinity. Deletion of an insert in the catalytic domain of PP7, predicted to function as a calmodulin-binding site, greatly decreased PP7 binding to calmodulin. These findings provide the first evidence for a plant protein phosphatase directly interacting with calmodulin and indicate that PP7 might be regulated by Ca(2+) levels in vivo.

Noncompetitive inhibition of plant protein Ser/Thr phosphatase PP7 by phosphate.

Changes in the cytoplasmic inorganic phosphate (P(i)) concentrations are an important cue for the plant cells to regulate their metabolism and phosphate homeostasis. However, phosphate sensors/receptors involved in this regulation are largely unknown. P(i) is a common nonspecific competitive inhibitor of phosphatases, usually in millimolar range. Here we report a procedure to refold recombinant Arabidopsis thaliana protein Ser/Thr phosphatase PP7 and demonstrate that PP7 is inhibited by submillimolar P(i) concentrations (IC(50) = 0.66 +/- 0.14 mM) via a mainly noncompetitive mechanism. The results indicate that PP7 may possess a specific P(i)-binding site responsible for its allosteric regulation, and suggest a possible phosphate sensor function for this protein phosphatase.

Organization of transport from endoplasmic reticulum to Golgi in higher plants.

In plant cells, the organization of the Golgi apparatus and its interrelationships with the endoplasmic reticulum differ from those in mammalian and yeast cells. Endoplasmic reticulum and Golgi apparatus can now be visualized in plant cells in vivo with green fluorescent protein (GFP) specifically directed to these compartments. This makes it possible to study the dynamics of the membrane transport between these two organelles in the living cells. The GFP approach, in conjunction with a considerable volume of data about proteins participating in the transport between endoplasmic reticulum and Golgi in yeast and mammalian cells and the identification of their putative plant homologues, should allow the establishment of an experimental model in which to test the involvement of the candidate proteins in plants. As a first step towards the development of such a system, we are using Sar1, a small G-protein necessary for vesicle budding from the endoplasmic reticulum. This work has demonstrated that the introduction of Sar1 mutants blocks the transport from endoplasmic reticulum to Golgi in vivo in tobacco leaf epidermal cells and has therefore confirmed the feasibility of this approach to test the function of other proteins that are presumably involved in this step of endomembrane trafficking in plant cells.

RdgC/PP5-related phosphatases: novel components in signal transduction.

A great variety of cellular functions are regulated by protein serine/threonine phosphatases (PP). This review summarises the current knowledge of the structural features, patterns of expression and involvement in signal transduction pathways of protein serine/threonine phosphatases related to PP5 and RdgC. Designated now as PP5/RdgC subfamily by P. T. W. Cohen in her 1997 study published in Trends in Biochemical Sciences, (Vol. 22, pp. 245-251), this heterogeneous group comprises phosphatases PP5/PPT, containing regulatory domains with tetratricopeptide repeats, RdgC/PPEF, which possess Ca2+-binding EF hand-type sites, and, recently discovered in plants, PP7. PP5 is ubiquitously expressed and appears to be a multifunctional phosphatase involved in a number of different signalling pathways. In contrast, expression of RdgC/PPEF phosphatases and PP7 is confined primarily to specialised sensory cells in animals and plants, respectively, which may be indicative of their more specialised roles in sensory signal transduction.

Non-classical protein Ser/Thr phosphatases: what are they for?

Protein serine/threonine phosphatases are involved in regulation of diverse cellular functions. This review is devoted to a novel group of protein Ser/Thr phosphatases, rdgC/PP5, which has been recently discovered in animals, fungi, and plants. Their structure, location, and possible functions are discussed.

Expression and characterization of PP7, a novel plant protein Ser/Thr phosphatase distantly related to RdgC/PPEF and PP5.

We have recently identified an Arabidopsis thaliana cDNA encoding a putative protein Ser/Thr phosphatase PP7, not closely related to any protein phosphatases in animals or fungi. Here, we describe the characterization of PP7 expressed in a bacterial system. The recombinant protein was inactive unless the longest insert in its catalytic domain was cleaved, suggesting that this insert is an autoinhibitory region. PP7 was resistant to okadaic acid, calyculin and fumonisin B1, and was stimulated by Mn2+ or Fe2+, while Ni2+ and Zn2+ were inhibitory. Polylysine stimulated PP7 activity towards p-nitrophenylphosphate but inhibited activity towards the most efficient protein substrate, myelin basic protein. A tentative model of the control of PP7 activity is proposed.

PP7, a plant phosphatase representing a novel evolutionary branch of eukaryotic protein Ser/Thr phosphatases.

We describe a novel protein Ser/Thr phosphatase from Arabidopsis thaliana, PP7, which is only 27-32% identical in amino acid sequence to the known phosphatases and is the most divergent member of the PPP (PP1/2A/2B) family for today. Some structural features suggest more close relationship of PP7 to the PP5/rdgC subfamily. PP7 contains all of the residues essential for the phosphatase activity and possesses three major insertions in its presumable C-terminal subdomain, which suggest its unique regulation and/or optimisation of its structure for interaction with specific substrates or regulators. A phosphatase structurally related to PP7 is expressed in rice. PP7 conservation between mono- and dicotyledonous plants may point to its essential role in the plant cell.

Proteins involved in membrane transport between the ER and the Golgi apparatus: 21 putative plant homologues revealed by dbEST searching.

Numerous proteins have been identified in yeast and mammalian cells which are involved in trafficking between the endoplasmic reticulum and the Golgi apparatus. A great number of partial cDNA sequences now available from the two major plant model species, Arabidopsis thaliana and Oryza sativa, makes it possible to identify putative plant homologues of known genes/proteins from non-plant species. The authors used this approach to screen the database of Expressed Sequence Tags (dbEST) in order to detect plant homologues of proteins involved in membrane transport between ER and Golgi. Availability of these partial sequences will facilitate the screening of cDNA and genomic libraries otherwise performed using heterologous probes derived from animal and yeast genes. As the plant Golgi complex differs in many respects from its mammalian and yeast counterparts, the dbEST clones found can be directly used for various functional assays (immunoprecipitation, two-hybrid analysis, transgenic plants etc.) to test the exact roles of the encoded proteins and identify their functional partners, some of which may be specific for plants.

[Ser/Thr-phosphatases from bovine retina: detection of cDNA coding for the catalytic subunit of the gamma-isoform PP2B and two homologs of rdgC/PPEF]. / Ser/Thr-fosfatazy setchatki byka: obnaruzhenie kDNK, kodiruiushchikh kataliticheskuiu sub''edinitsu gamma-izoformy PP2B i dva gomologa rdgC/PPEF.

The expression of genes for Ser/Thr phosphatases of the PP1/2A/2B class in the bovine retina was analyzed using PCR on cDNA. The mRNAs were found for the following phosphatases: PP1 (alpha- and gamma-isoforms), PP2A (alpha- and beta-isoforms), PPV, and PP2B (beta- and gamma-isoforms). Earlier, PP2B gamma-isoform was considered testis-specific. cDNAs of two phosphatases related to rdgC/PPEF were also detected. Thus, there are mRNAs of at least four phosphatases in retina that may be regulated by Ca2+.

[Genes of plant Ser/Thr protein phosphatases: detection of sequences related to PPT/rdgC]. / Geny Ser/Thr-fosfataz rasteni'i: detektsiia posledovatel'noste'i, rodstvennykh PPT/rdgC.

An unknown sequence that may encode a fragment of the Ser/Thr protein phosphatase (designated PP6Zm) related to PPT/rdgC phosphatases was identified using PCR on maize genomic DNA. A dbEST search using a partial amino acid sequence of PP6Zm revealed a putative homolog of PP6Zm expressed in Arabidopsis thaliana (EMBL AT6726). A search of the SwissProt database indicated that the partial amino acid sequence of AT6726 has the highest identity (54.3%) to the rdgC phosphatase from Drosophila melanogaster. The maize phosphatase PP1Zm6, described previously as a PP1 isoform (EMBO J., 1993, vol. 12, p. 3497), was found by us to be plant homolog of mammalian PPT. In addition, six fragments of new (pseudo) genes homologous to the phosphatase genes encoding PP1, PP2A, and PPX isoforms were detected in the maize genome. The existence in maize of a multigene PP2A family, reported only for dicotyledons, and of a PP1 multigene family, found earlier in both di- and monocotyledons, was shown.

Calcium-activated opsin phosphatase activity in retinal rod outer segments.

We describe the presence in bovine retinal rod outer segments of a phosphatase which dephosphorylates phosphoopsin with an efficiency similar to that of PP2A, and which is stimulated by submicromolar levels of Ca2+ (half-maximal activation, 0.4-0.5 microM). This enzyme is designated CA2+ -activated opsin phosphatase (CAOP). CAOP has a molecular mass of 70-75 kDa as determined by gel filtration on Superose 12 and exhibits reversible Ca2+ -dependent oligomerization. An unidentified protein of approximately 25 kDa is necessary for full activity of CAOP and for cooperative binding of Ca2+ (h > 2). CAOP does not require Mg2+ and is inhibited by okadaic acid (median inhibitory concentration > 25 microM), which suggests that it is related to the PP1/2A/2b class of protein phosphatases. Like PP2B, CAOP is inhibited by trifluoperazine (median inhibitory concentration 40 microM), but calmodulin has no effect on CAOP activity, and CAOP is inhibited by mastoparan at much higher concentrations than PP2b. This combination of properties suggests that CAOP is not identical to any characterized protein phosphatase. Since the cytoplasmic concentration of Ca2+ -sensitive opsin phosphatase activity suggests that light-dependent Ca2+ levels may control rhodopsin dephosphorylation.

Conformation of surface exposed N-terminus part of bacteriorhodopsin studied by transferred NOE technique.

Interaction of the monoclonal antibody A5 raised against native bacteriorhodopsin (BR) with the synthetic peptide pGlu1-Ala-Gln-Ile-Thr-Gly-Arg7-NH2, corresponding to the amino acid sequence 1-7 was studied by transferred nuclear Overhauser effect (TRNOE) spectroscopy. The denaturing reagents and the specially designed pulse sequences which eliminate broad signals from the TRNOE spectra were used to favour evaluation of the TRNOE peaks. On the basis of the data obtained, the conformation of peptide bound with A5 was calculated. A model of the mutual arrangement of bacteriorhodopsin N-terminus and the first transmembrane alpha-helical segment 8-32 was proposed.

[p26--a calcium binding protein from photoreceptor cells in the bovine retina: primary structure and expression in E. coli]. / p26--kal'tsiisviazyvaiushchii belok fotoretseptornykh kletok setchatki byka: pervichnaia struktura i ékspressiia v E. coli.

The primary structure of the bovine retinal calcium binding protein P26 has been determined by the parallel analysis of the protein and the corresponding cDNA. This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina. P26 was expressed in E. coli as a fusion protein and, after purification by affinity chromatography on IgG-Sepharose 6, cleaved off with enteropeptidase.

P26-calcium binding protein from bovine retinal photoreceptor cells.

The primary structure of bovine retinal calcium binding protein P26 has been determined by parallel analysis of protein and corresponding cDNA. This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina. Preliminary data are presented on expression of P26 as a fusion protein in E. coli.

Functioning of quinone acceptors in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus.

The photosynthetic reaction centers (RC) of the green bacterium Chloroflexus aurantiacus have been investigated by spectral and electrometrical methods. In these reaction centers, the secondary quinone was found to be reconstituted by the addition of ubiquinone-10. The equilibrium constant of electron transfer between primary (QA) and secondary (QB) quinones was much higher than that in RC of purple bacteria. The QB binding to the protein decreased under alkalinization with apparent pK 8.8. The single flash-induced electric responses were about 200 mV. An additional electrogenic phase due to the QB protonation was observed after the second flash in the presence of exogenous electron donors. The magnitude of this phase was 18% of that related to the primary dipole (P+QA-) formation. Since the C. aurantiacus RC lacks H-subunit, this subunit was not an obligatory component for electrogenic QB protonation.

[Study of the structure of the photosynthetic reaction center of the green thermophilic bacteria Chloroflexus aurantiacus]. / Izuchenie structury fotosinteticheskogo reaktsionnogo tsentra zelenoi termofil'noi bakterii Chloroflexus aurantlacus.

Analysis of the Chloroflexus aurantiacus reaction centre (RC) using both protein and recombinant DNA techniques resulted in determination of its polypeptide composition and the primary structures of its two subunits. A model of the polypeptide chains' folding in the membrane is suggested based on: i) homology between L- and M-subunits of Chloroflexus aurantiacus RC and their counterparts in purple bacteria; ii) comparison of their hydropathy plots, and iii) data on the tertiary structures of purple bacteria RCs. The role of a number of functionally important amino acid residues in the RC electron transport activity is discussed. Limited proteolysis of the RC under non-denaturing conditions was used to determine the contribution of the N-terminal regions to its thermal stability.