Structural basis for the thermostability of ferredoxin from the cyanobacterium Mastigocladus laminosus

Plant-type ferredoxins (Fds) carry a single [2Fe-2S] cluster and serve as electron acceptors of photosystem I (PSI). The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus displays optimal activity at 65 degrees C. In order to reveal the molecular factors that confer thermostability, the crystal structure of M.laminosus Fd (mFd) was determined to 1.25 A resolution and subsequently analyzed in comparison with four similar plant-type mesophilic ferredoxins. The topologies of the plant-type ferredoxins are similar, yet two structural determinants were identified that may account for differences in thermostability, a salt bridge network in the C-terminal region, and the flexible L1,2 loop that increases hydrophobic accessible surface area. These conclusions were verified by three mutations, i.e. substitution of L1,2 into a rigid beta-turn ((Delta)L1,2) and two point mutations (E90S and E96S) that disrupt the salt bridge network at the C-terminal region. All three mutants have shown reduced electron transfer (ET) capabilities and [2Fe-2S] stability at high temperatures in comparison to the wild-type mFd. The results have also provided new insights into the involvement of the L1,2 loop in the Fd interactions with its electron donor, the PSI complex.     

ZNF750 Is Expressed in Differentiated Keratinocytes and Regulates Epidermal Late Differentiation Genes

Disrupted skin barrier due to altered keratinocyte differentiation is common in pathologic conditions such as atopic dermatitis, ichthyosis and psoriasis. However, the molecular cascades governing keratinocyte terminal differentiation are poorly understood. We have previously demonstrated that a dominant mutation in ZNF750 leads to a clinical phenotype reminiscent of psoriasis and seborrheic dermatitis. Here we show that ZNF750 is a nuclear protein bearing a functional C-terminal nuclear localization signal. ZNF750 was specifically expressed in the epidermal suprabasal layers and its expression was augmented during differentiation, both in human skin and in-vitro, peaking in the granular layer. Silencing of ZNF750 in Ca2+-induced HaCaT keratinocytes led to morphologically apparent arrest in the progression of late differentiation, as well as diminished apoptosis and sustained proliferation. ZNF750 knockdown cells presented with markedly reduced expression of epidermal late differentiation markers, including gene subsets of epidermal differentiation complex and skin barrier formation such as FLG, LOR, SPINK5, ALOX12B and DSG1, known to be mutated in various human skin diseases. Furthermore, overexpression of ZNF750 in undifferentiated cells induced terminal differentiation genes. Thus, ZNF750 is a regulator of keratinocyte terminal differentiation and with its downstream targets can serve in future elucidation of therapeutics for common diseases of skin barrier.     

CHRD, a plant member of the evolutionarily conserved YjgF family, influences photosynthesis and chromoplastogenesis

Studies on the carotenoid-overaccumulating structures in chromoplasts have led to the characterization of proteins termed plastid lipid-associated proteins (PAPs), involved in the sequestration of hydrophobic compounds. Here we characterize the PAP CHRD, which, based on sequence homology, belongs to a highly conserved group of proteins, YER057c/YjgF/UK114, involved in the regulation of basic and vital cellular processes in bacteria, yeast and animals. Two nuclear genes were characterized in tomato plants: one (LeChrDc) is constitutively expressed in various tissues and the other (LeChrDi) is induced by stress in leaves and is upregulated by developmental cues in floral tissues. Using RNAi and antisense approaches, we show their involvement in biologically significant processes such as photosynthesis. The quantum yield of photosynthetic electron flow in transgenic tomato leaves with suppressed LeChrDi/c expression was 30–50% of their control, non-transgenic counterparts and was ascribed to lower PSI activity. Transgenic flowers with suppressed LeChrDi/c also accumulated up to 30% less carotenoids per unit protein as compared to control plants, indicating an interrelationship between PAPs and floral-specific carotenoid accumulation in chromoplasts. We suggest that CHRD’s role in the angiosperm reproductive unit may be a rather recent evolutionary development; its original function may have been to protect the plant under stress conditions by preserving plastid functionality.Y. Leitner-Dagan and M. Ovadis contributed equally to this work     

Impact of PsbTc on forward and back electron flow, assembly, and phosphorylation patterns of photosystem II in tobacco

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn₄Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth. Mutant plants appear normal green, and levels of PSII proteins are not affected. Yet, PSII-dependent electron transport, stability of PSII dimers, and assembly of PSII light-harvesting complexes (LHCII) are significantly impaired. PSII light sensitivity is moderately increased and recovery from photoinhibition is delayed, leading to faster D1 degradation in ΔpsbTc under high light. Thermoluminescence emission measurements revealed alterations of midpoint potentials of primary/secondary electron-accepting plastoquinone of PSII interaction. Only traces of CP43 and no D1/D2 proteins are phosphorylated, presumably due to structural changes of PSII in ΔpsbTc. In striking contrast to the wild type, LHCII in the mutant is phosphorylated in darkness, consistent with its association with PSI, indicating an increased pool of reduced plastoquinone in the dark. Finally, our data suggest that the secondary electron-accepting plastoquinone of PSII site, the properties of which are altered in ΔpsbTc, is required for oxidation of reduced plastoquinone in darkness in an oxygen-dependent manner. These data present novel aspects of plastoquinone redox regulation, chlororespiration, and redox control of LHCII phosphorylation.     

Biogenesis of chloroplast membranes. I. Plastid dedifferentiation in a dark-grown algal mutant (Chlamydomonas reinhardi)

This paper describes the morphology and photosynthetic activity of a mutant of Chlamydomonas reinhardi (y-1) which is unable to synthesize chlorophyll in the dark. When grown heterotrophically in the light, the mutant is indistinguishable from the wild type Chlamydomonas. When grown in the dark, chlorophyll is diluted through cell division and the photosynthetic activity (oxygen evolution, Hill reaction, and photoreduction of NADP) decays at a rate equal to or faster than that of chlorophyll dilution. However, soluble enzymes associated with the photosynthetic process (alkaline FDPase, NADP-linked G-3-P dehydrogenase, RuDP carboxylase), as well as cytochrome f and ferredoxin, continue to be present in relatively high concentrations. The enzymes involved in the synthesis of the characteristic lipids of the chloroplast (including mono- and digalactoside glycerides, phosphatidyl glycerol, and sulfolipid) are still detectable in dark-grown cells. Such cells accumulate large amounts of starch granules in their plastids. On onset of illumination, dark-grown cells synthesize chlorophyll rapidly, utilizing their starch reserve in the process. At the morphological level, it was observed that during growth in the dark the chloroplast lamellar system is gradually disorganized and drastically decreased in extent, while other subchloroplast components are either unaffected (pyrenoid and its tubular system, matrix) or much less affected (eyespot, ribosomes). It is concluded that the dark-grown mutant possesses a partially differentiated plastid and the enzymic apparatus necessary for the synthesis of the chloroplast membranes (discs). The advantage provided by such a system for the study of the biogenesis of the chloroplast photosynthetic membranes is discussed.     

Gene expression and the concept of the phenotype

While the definition of the 'genotype' has undergone dramatic changes in the transition from classical to molecular genetics, the definition of the 'phenotype' has remained for a long time within the classical framework. In addition, while the notion of the genotype has received significant attention from philosophers of biology, the notion of the phenotype has not. Recent developments in the technology of measuring gene-expression levels have made it possible to conceive of phenotypic traits in terms of levels of gene expression. We demonstrate that not only has this become possible but it has also become an actual practice. This suggests a significant change in our conception of the phenotype: as in the case of the 'genotype', phenotypes can now be conceived in quantitative and measurable terms on a comprehensive molecular level. We discuss in what sense gene expression profiles can be regarded as phenotypic traits and whether these traits are better described as a novel concept of phenotype or as an extension of the classical concept. We argue for an extension of the classical concept and call for an examination of the type of extension involved.     

Phosphorylation of chlamydomonas reinhardi chloroplast membrane proteins in vivo and in vitro

Phosphorylation of thylakoid membrane proteins in the chloroplast of wild-type and mutant strains of Chlamydomonas reinhardi has been studied in vivo and in vitro. Intact cells or purified membranes were labeled with [32P]orthophosphate or [gamma-32P]ATP, respectively, and the presence of phosphorylated polypeptides was detected by autoradiography after membrane fractionation by SDS PAGE. The 32P was esterified to serine and threonine residues. At least six polypeptides were phosphorylated in vitro and in vivo, and corresponded to components of the photosystem II complex contributing to the formation of the light-harvesting-chlorophyll (LHC) a,b-protein complex, the DCMU binding site (32-35 kdaltons), and the reaction center (26 kdaltons). In agreement with previous reports (Alfonzo, et al., 1979, Plant Physiol., 65:730-734; and Bennett, 1979, FEBS (Fed. Eur. Biochem. Soc.) Lett., 103:342-344), the membrane-bound protein kinase was markedly stimulated by light in vitro via a mechanism requiring photosystem II activity. Phosphorylation of thylakoid membrane polypeptides in vivo was, however, completely independent of illumination. Similar amounts of phosphate were incorporated into the photosynthetic membranes of cells incubated in the dark, in white light with or without 3-(3,4- dichlorophenyl-1,1-dimethyl urea (DCMU), or in red or far-red light. Different turnovers of the phosphate were observed in the light and dark, and a phosphoprotein phosphatase involved in this turnover process was also associated with the membrane. Comparison of the amount of esterified phosphate per protein in vivo and the maximum incorporation in isolated membranes revealed that only a small fraction of the available sites could be phosphorylated in vitro. In contrast to the DCMU binding site, the LHC and 26-kdalton polypeptide were not phosphorylated in vivo when the reaction center II polypeptides of 44- 54 kdaltons were missing. The finding that all the phosphoproteins appear to be components of the photosystem II complex and are only partially dephosphorylated in vivo suggests strongly that protein phosphorylation might play an important role in the maintenance of the organizational integrity of this complex. The observation that the LHC is not phosphorylated in the absence of the reaction center lends support to this idea.     

Induction of ATP depletion, intramembrane particle aggregation and exposure of membrane phospholipids in chicken erythrocytes by local anesthetics and tranquilizers

Incubation of chicken erythrocytes with 1 mM tetracaine, 10 mM lidocaine and 0.24–0.48 mM chlorpromazine significantly reduced the ATP content of the cells, while procaine even at concentrations as high as 10 mM had only a slight effect. When chlorpromazine was used, it was found that the final level of the ATP was dependent on the drug concentration, which at 0.48 mM depletes the cells to about 10% of the initial ATP content. The ATP depletion of chicken erythrocytes was accompanied by dephosphorylation of certain membrane proteins which were identified by acrylamide gel electrophoresis as an 180 000 dalton protein band and peptides with molecular weight of 60 000–100 000. Treatment of chicken and rat erythrocytes with 0.5 mM tetracaine and 1 mM lidocaine or with 0.48 mM chlorpromazine induced significant aggregation of intramembrane particles as revealed by the freeze-etching technique. Procaine (10 mM) had no effect. Incubation of chicken erythrocytes with the above-mentioned drugs induced also exposure of the masked membrane phospholipids to the action of phospholipase-C (Bacillus cereus) and to phospholipase A₂ (bee venom). Negligible amounts of phospholipids were hydrolyzed in the untreated cells, while about 40% of the membrane phosphatidylethanolamine and 50% of the phosphatidylcholine were hydrolyzed by phospholipase A₂ in chicken erythrocytes treated with 0.48 mM chlorpromazine.Treatment of chicken and rat erythrocytes with 0.48 mM chlorpromazine resulted also in an increase in the amount of the phospholipid fraction which could be extracted by dry ether. About 41% and 60% of phospholipids were respectively, as compared to 25% and 35% of phospholipids extracted from the same untreated cells.     

Hyperchlorhidrosis caused by homozygous mutation in CA12, encoding carbonic anhydrase XII

Excessive chloride secretion in sweat (hyperchlorhidrosis), leading to a positive sweat test, is most commonly indicative of cystic fibrosis yet is found also in conjunction with various metabolic, endocrine, and dermatological disorders. There is conflicting evidence regarding the existence of autosomal-recessive hyperchlorhidrosis. We now describe a consanguineous Israeli Bedouin kindred with autosomal-recessive hyperchlohidrosis whose sole symptoms are visible salt precipitates after sweating, a preponderance to hyponatremic dehydration, and poor feeding and slow weight gain at infancy. Through genome-wide linkage analysis, we demonstrate that the phenotype is due to a homozygous mutation in CA12, encoding carbonic anhydrase XII. The mutant (c.427G>A [p.Glu143Lys]) protein showed 71% activity of the wild-type enzyme for catalyzing the CO₂ hydration to bicarbonate and H⁺, and it bound the clinically used sulfonamide inhibitor acetazolamide with high affinity (K_I of 10 nM). Unlike the wild-type enzyme, which is not inhibited by chloride, bromide, or iodide (K_Is of 73–215 mM), the mutant is inhibited in the submicromolar range by these anions (K_Is of 0.37–0.73 mM).