The integration of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membrane proceeds in two steps. First, LHCP interacts with a chloroplast signal recognition particle (cpSRP) to form a soluble targeting intermediate called the transit complex. Second, LHCP integrates into the thylakoid membrane in the presence of GTP, at least one other soluble factor, and undefined membrane components. We previously determined that cpSRP is composed of 43-and 54-kDa polypeptides. We have examined the subunit stoichiometry of cpSRP and find that it is trimeric and composed of two subunits of cpSRP43/subunit of cpSRP54. A chloroplast homologue of FtsY, an Escherichia coli protein that is critical for the function of E. coli SRP, was found largely in the stroma unassociated with cpSRP. When chloroplast FtsY was combined with cpSRP and GTP, the three factors promoted efficient LHCP integration into thylakoid membranes in the absence of stroma, demonstrating that they are all required for reconstituting the soluble phase of LHCP transport. SRP1 mediates the cotranslational targeting of endomembrane and secretory proteins to the endoplasmic reticulum in eukaryotes and of polytopic membrane proteins to the cytoplasmic membrane in prokaryotes (1-3). Cytosolic forms of SRP are ubiquitous in eukaryotic and prokaryotic organisms. All contain, at a minimum, a 54-kDa GTPase subunit and an RNA (1, 2). Membrane targeting is facilitated by an interaction between SRP and an SRP receptor (4 -6). In eukaryotes, the receptor consists of two GTPases, a peripheral protein (the SRP receptor ␣-subunit), and an integral membrane polypeptide (the SRP receptor ␤-subunit) (7,8). The localization of the SRP receptor to the membrane may facilitate, but is not essential for, targeting (9). A key feature of the SRP/SRP receptor interaction is the ability of the SRP receptor ␣-subunit and SRP54 to reciprocally stimulate their GTP hydrolysis activities upon mutual binding in the presence of SRP RNA and thereby to regulate the GTP hydrolysis cycle associated with SRP-dependent protein targeting (10, 11).Recently, a specialized SRP was found in the chloroplast (12, 13). cpSRP contains a homologue of SRP54 (14), but differs from cytoplasmic forms, as it lacks an RNA, contains a novel 43-kDa subunit, and interacts with substrates post-translationally (12,15,16). Both genetic and biochemical evidence indicates that the 43-kDa subunit is essential for this posttranslational interaction (12,15,17). The known substrates of cpSRP are the LHCPs, hydrophobic proteins that are synthesized in the cytoplasm and are post-translationally transported to the internal membranes of the chloroplast via the soluble phase (18,19). The solubility of LHCP is maintained in the stroma by its binding to cpSRP to form the targeting intermediate termed the transit complex (12,16,20).The transit complex can be reconstituted in vitro from purified cpSRP and LHCP, suggesting that it is composed of cpSRP54, cpSRP43, and LHCP. However, one unresolved issue is the subunit stoich...

A sensor histidine kinase of Synechococcus sp. strain PCC7942, designated nblS, was previously identified and shown to be critical for the acclimation of cells to high-light and nutrient limitation conditions and to influence the expression of a number of light-responsive genes. The nblS orthologue in Synechocystis sp. strain PCC6803 is designated dspA (also called hik33). We have generated a dspA null mutant and analyzed global gene expression in both the mutant and wild-type strains under high-and low-light conditions. The mutant is aberrant for the expression of many genes encoding proteins critical for photosynthesis, phosphate and carbon acquisition, and the amelioration of stress conditions. Furthermore, transcripts from a number of genes normally detected only during exposure of wild-type cells to high-light conditions become partially constitutive in the low-light-grown dspA mutant. Other genes for which transcripts decline upon exposure of wild-type cells to high light are already lower in the mutant during growth in low light. These results suggest that DspA may influence gene expression in both a positive and a negative manner and that the dspA mutant behaves as if it were experiencing stress conditions (e.g., high-light exposure) even when maintained at near-optimal growth conditions for wild-type cells. This is discussed with respect to the importance of DspA for regulating the responses of the cell to environmental cues.Photosynthetic organisms have evolved intricate mechanisms for sensing and acclimating to environmental change. Parameters such as light quality, light intensity, and nutrient availability can modulate both the structure and the function of the photosynthetic machinery. Physiological and biochemical changes elicited by external cues include modification of lightharvesting complex (LHC) synthesis and degradation (6,12,27,29,38,45,65,75), changes in absorption and excitation energy transfer properties of LHC (11,18,28,59), and a modification of reaction center function (59, 80). Intracellular cues critical for controlling cellular processes during acclimation may reflect the cell's growth potential, cellular redox conditions, and/or accumulation of reactive oxygen species (52,77).Precise control over the fate of absorbed excitation energy is critical for cell viability during exposure of photosynthetic cells to excess excitation, since energized pigment molecules may trigger the production of damaging, reactive oxygen species (2). Over the short term, cells can dissipate excess absorbed excitation energy as heat by quenching excited pigment molecules in the LHC or by eliciting a state transition in which the LHC of photosystem II (PS II) directs its excitation energy to PS I, where quenching can occur. Over the long term, excess excitation may cause a dramatic reduction in the level of LHC. In cyanobacteria, a reduction in LHC size is reflected in reduced levels of transcripts encoding light-absorbing polypeptides (or phycobiliproteins) of the major LHC (or phycobilisomes). Several st...

Tomatoes (Lycopersicon esculentum) express two forms of leucine aminopeptidase (LAP-A and LAP-N) and two LAP-like proteins. The relatedness of LAP-N and LAP-A was determined using affinity-purified antibodies to four LAP-A protein domains. Antibodies to epitopes in the most N-terminal region were able to discriminate between LAP-A and LAP-N, whereas antibodies recognizing central and COOH-terminal regions recognized both LAP polypeptides. Two-dimensional immunoblots showed that LAP-N and the LAP-like proteins were detected in all vegetative (leaves, stems, roots, and cotyledons) and reproductive (pistils, sepals, petals, stamens, and floral buds) organs examined, whereas LAP-A exhibited a distinct expression program. LapN was a single-copy gene encoding a rare-class transcript. A full-length LapN cDNA clone was isolated, and the deduced sequence had 77% peptide sequence identity with the wound-induced LAP-A. Leucyl aminopeptidases (LAPs; EC 3.4.11.1) are members of the M17 family of peptidases (Barrett et al., 1998). LAPs are ubiquitous being found in animals, plants, and prokaryotic cells. These hexameric metallopeptidases catalyze the release of the N-terminal residues from protein, peptide, fluorometric, and chromogenic substrates. The best characterized LAPs are from Bos taurus, Escherichia coli and tomato (Lycopersicon esculentum). X-ray crystal structures of the bovine and E. coli LAPs have provided insight into the LAP catalytic mechanism (Kim and Lipscomb, 1994;Sträter and Lipscomb, 1995;Sträter et al., 1999a). The roles of selected residues of the E. coli and tomato LAPs in chromogenic or peptide substrate catalysis, respectively, have been tested by site-directed mutagenesis (Sträter et al., 1999b;Gu and Walling, 2002).In most plants, three classes of LAP-related polypeptides are detected using a tomato LAP antiserum, including the 66-and 77-kD LAP-like proteins and the 55-kD neutral LAP (LAP-N; Chao et al., 2000). Only in a subset of the Solanaceae is a second 55-kD LAP species (LAP-A) detected (Hildmann et al., 1992;Gu et al., 1996b;Chao et al., 2000). In tomato, LAP-A protomers have an acidic pI and are encoded by two genes (LapA1 and LapA2), which are expressed during floral and fruit development. The LapA genes are not expressed in foliage from healthy plants (Chao et al., 1999). However, LapA RNAs, proteins, and activities accumulate locally and systemically in leaves after wounding, Pseudomonas syringae pv. tomato and Phytophthora parasitica infection, and caterpillar feeding (Pautot et al., 1993(Pautot et al., , 2001Gu et al., 1996b;Chao et al., 1999;Jwa and Walling, 2001). The activation of LapA gene expression by jasmonic acid (JA), abscisic acid, the phytotoxin coronatine (a JA mimic), and suppression of LapA by salicylic acid is consistent with the regulation of the tomato LapA genes by the wound octadecanoid pathway (Chao et al., 1999). LapA genes also respond to signals generated during water deficit and salinity stress (Chao et al., 1999). The potato (Solanum tuberosum) Lap RNAs also ac...

1 Fractalkine is a CX 3 C chemokine for mononuclear leukocytes that is expressed mainly by vascular cells, and regulated by pro-in¯ammatory cytokines. This study investigated signal transduction mechanisms by which tumor necrosis factor (TNF)-a stimulated fractalkine expression in cultured rat vascular smooth muscle cells (VSMCs), and the modulatory eect of a haemorrheologic agent, pentoxifylline, on its production. 2 TNF-a (1 ± 50 ng ml 71 ) stimulated fractalkine mRNA and protein expression in concentrationand time-dependent manners. Pretreatment with calphostin C (0.4 mM, a selective inhibitor of protein kinase C (PKC), and PD98059 (40 mM), a speci®c inhibitor of p42/44 mitogen-activated protein kinase (MAPK) kinase, attenuated TNF-a-stimulated fractalkine mRNA and protein expression. In contrast, H-89 (2 mM), a selective inhibitor of cAMP-dependent protein kinase, wortmannin (0.5 mM), a selective inhibitor of phosphatidylinositol 3-kinase, and SB203580 (40 mM), a speci®c inhibitor of p38 MAPK, had no discernible eect. 3 The ubiquitin/proteosome inhibitors, MG132 (10 mM) and pyrrolidine dithiocarbamate (200 mM), suppressed activation of NF-kB as well as stimulation of fractalkine mRNA and protein expression by TNF-a. 4 TNF-a-activated phosphorylation of PKC was blocked by calphostin C, whereas TNF-aaugmented phospho-p42/44 MAPK and phospho-c-Jun levels were reduced by PD98059. Neither calphostin C nor PD98059 aected TNF-a-induced degradation of I-kBa or p65 nuclear translocation. 5 Pretreatment with pentoxifylline (0.1 ± 1 mg ml 71) decreased TNF-a-stimulated fractalkine mRNA and protein expression, which was preceded by a reduction in TNF-a-activated phosphorylation of PKC, p42/44 MAPK and c-Jun as well as degradation of I-kBa and p65/NFkB nuclear translocation. 6 These data indicate that activation of PKC, p42/44 MAPK kinase, and NF-kB are involved in TNF-a-stimulated fractalkine production in VSMCs. Down-regulation of the PKC, p42/44 MAPK, and p65/NF-kB signals by PTX may be therapeutically relevant and provide an explanation for the anti-fractalkine eect of this drug.

Cyanobacteria constitute an ancient, diverse and ecologically important bacterial group. The responses of these organisms to light and nutrient conditions are finely controlled, enabling the cells to survive a range of environmental conditions. In particular, it is important to understand how cyanobacteria acclimate to the absorption of excess excitation energy and how stress-associated transcripts accumulate following transfer of cells from low-to high-intensity light. In this study, quantitative RT-PCR was used to monitor changes in levels of transcripts encoding chaperones and stress-associated proteases in three cyanobacterial strains that inhabit different ecological niches: the freshwater strain Synechocystis sp. PCC 6803, the marine high-light-adapted strain Prochlorococcus MED4 and the marine low-light-adapted strain Prochlorococcus MIT9313. Levels of transcripts encoding stress-associated proteins were very sensitive to changes in light intensity in all of these organisms, although there were significant differences in the degree and kinetics of transcript accumulation. A specific set of genes that seemed to be associated with high-light adaptation (groEL/groES, dnaK2, dnaJ3, clpB1 and clpP1) could be targeted for more detailed studies in the future. Furthermore, the strongest responses were observed in Prochlorococcus MED4, a strain characteristic of the open ocean surface layer, where hsp genes could play a critical role in cell survival.

The constitutive and wound-inducible leucine aminopeptidases (LAP-N and LAP-A, respectively) of tomato encode 60-kDa proteins with 5-kDa presequences that resemble chloroplast-targeting peptides. Cell fractionation studies and immunoblot analyses of chloroplast and total proteins have suggested a dual location of the mature LAP-A proteins in the cytosol and the plastids. In this study, the subcellular localization of tomato LAPs was further investigated using in vitro chloroplast-targeting assays and immunocytochemical techniques at the light and TEM levels. In vitro-translated LAP-A1 and LAP-N preproteins were readily transported into pea chloroplasts and processed into mature proteins of 55 kDa indicating the presence of a functional chloroplast-targeting signal in the LAP-A1 and LAP-N protein precursors. In addition, a LAP polyclonal and a LAP-A-specific antisera were used to immunolocalize LAP proteins in leaves from healthy, wounded and methyl jasmonate (MeJA)-treated plants. Low levels of LAPs and/or LAP-like proteins were detected in leaves from unwounded plants. The LAP polyclonal antiserum, which detected LAP-A, LAP-N and LAP-like proteins, and the LAP-A specific antibodies, which detected only LAP-A, showed that LAP levels increased in leaf sections after wounding and MeJA treatments. LAP-A proteins were primarily detected within the chloroplasts of spongy and palisade mesophyll cells. The localization of LAP-A was distinct from the location of early wound-response proteins that are important in the biosynthesis of jasmonic acid or systemin and more similar to the late wound-response proteins with primary roles in defense. The importance of these findings relative to the potential roles of LAP-A in defense is discussed.

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Type I interferon induced MxA has antiviral activity against RNA viruses. Here we demonstrate a previously unrecognized MxA isoform induced by herpes simplex virus‐1 (HSV‐1) in human fibroblasts. Transcript encoding MxA splice variant has a deletion in Exons 14–16 which encode a central interactive domain associated with recognition of viral nucleocapsids and is predicted to encode a novel peptide sequence at the C‐terminal region as compared with IFN ‐induced MxA prototype. Polyclonal antiserum raised in rabbit against C‐terminal half of MxA splice variant demonstrated that HSV‐1 induced variant MxA undergoes nuclear translocation associated with viral replication compartment. Infection of variant MxA‐overexpressing fibroblasts with HSV‐1 resulted in the enhancement of viral yields but the yields were decreased in MxA‐knockdown cell by siRNA, suggesting that HSV‐1 induced MxA splice variant enhances viral replication. Both IFN‐ α and HSV‐1 are able to activate the promoter of MxA in luciferase reporter assay; however, only the MxA variant is translated in virus‐infected cells. Thus, differential expression of MxA controlled by IFN‐α or HSV‐1 could result in the increase of host resistance or favoring viral replication, depending on stimulating event. In summary, modulation of cellular mRNA could be a novel mechanism for human alphaherpesviruses to avoid innate immune detection.