Structural and functional insights into nucleocytoplasmic transport

The cell nucleus is surrounded by a double membrane system, the nuclear envelope (NE), with the outer nuclear membrane being continuous with the endoplasmic reticulum. Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes, forming aqueous channels that allow free diffusion of small molecules but that also mediate the energy-dependent transport of large macromolecules. The NPC represents the largest known molecular complex and is composed of about 30 different proteins, termed nucleoporins (Nups). Here, we review recent studies that provide novel insight into the structural and functional organization of nucleocytoplasmic transport. In addition, prospects towards a high resolution model of the nuclear pore are discussed.     

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.     

High CO2 concentration alleviates the block in photosynthetic electron transport in an ndhB-inactivated mutant of Synechococcus sp. PCC 7942.

The high-concentration CO2-requiring mutant N5 of Synechococcus sp. PCC 7942 was obtained by the insertion of a kanamycin-resistant gene at the EcoRI site, 12.4 kb upstream of rbc. The mutant is unable to accumulate inorganic carbon internally and exhibits very low apparent photosynthetic affinity for inorganic carbon but a photosynthetic Vmax similar to that of the wild type. Sequence and northern analyses showed that the insertion inactivated a gene highly homologous to ndhB, encoding subunit II of NADH dehydrogenase in Synechocystis sp. PCC 6803 (T. Ogawa [1991] Proc Natl Acad Sci USA 88: 4275-4279). When the mutant and the wild-type cells were exposed to 5% CO2 in air, their photosynthetic electron transfer capabilities, as revealed by fluorescence and thermoluminescence measurements, were similar. On the other hand, a significant decrease in variable fluorescence was observed when the mutant (but not the wild-type) cells were exposed to low CO2 under continuous light. The same treatment also resulted in a shift (from 38-27 degrees C) in the temperature at which the maximal thermoluminescence emission signal was obtained in the mutant but not in the wild type. These results may indicate that subunit II of NADH dehydrogenase is essential for the functional operation of the photosynthetic electron transport in Synechococcus under low but not high levels of CO2. We suggest that the inability to accumulate inorganic carbon under air conditions stems from disrupture of electron transport in this mutant.     

Thylakoid membrane energization and swelling in photoinhibited Chlamydomonas cells is prevented in mutants unable to perform cyclic electron flow

Photoinhibition of Photosystem II in unicellular algae in vivo is accompanied by thylakoid membrane energization and generation of a relatively high pH as demonstrated by ¹⁴C-methylamine uptake in intact cells. Presence of ammonium ions in the medium causes extensive swelling of the thylakoid membranes in photoinhibited Chlamydomonas reinhardtii but not in Scenedesmus obliquus wild type and LF-1 mutant cells. The rise in pH and the related thylakoid swelling do not occur at light intensities which do not induce photoinhibition. The rise in pH and membrane energization are not induced by photoinhibitory light in C. reinhardtii mutant cells possessing an active Photosystem II but lacking cytochrome b₆/f, plastocyanin or Photosystem I activity and thus being unable to perform cyclic electron flow around Photosystem I. In these mutants the light-induced turnover of the D1 protein of Reaction Center II is considerably reduced. The high light-dependent rise in pH is induced in the LF-1 mutant of Scenedesmus which can not oxidize water but otherwise possesses an active Reaction Center II indicating that PS II-linear electron flow activity and reduction of plastoquinone are not required for this process. Based on these results we conclude that photoinhibition of Photosystem II activates cyclic electron flow around Photosystem I which is responsible for the high membrane energization and pH rise in cells exposed to excessive light intensities.Abbreviations cyt b₆/f cytochrome b₆/f - Diuron 3-(3,4-dichlorophenyl)-1 dimethyl urea - Q_B the secondary quinone acceptor of reaction center II - DNP 2,4,Dinitrophenol - FCCP carbonyl cyanide trifluoromethoxy phenylhydrazone - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

Mechanism of photoinhibition in vivo. A reversible light-induced conformational change of reaction center II is related to an irreversible modification of the D1 protein

The light-induced inactivation of the photochemical reaction center II (RCII) of oxygenic chloroplasts (photoinhibition) was investigated in cells and isolated thylakoids of the green alga Chlamydomonas reinhardtii. The process is resolved into a reversible conformational change followed by an irreversible modification of RCII D1 protein. The light-induced changes in vivo persisted in isolated thylakoids. The first step is characterized by (i) destabilization of the secondary acceptor semiquinone anion, Q-B, bound to the D1 protein. This is demonstrated by a reduction in the activation energy of S2,3Q-B charge recombination as measured by the thermoluminescence technique; and (ii) a rise in the intrinsic fluorescence and a decrease of the maximal fluorescence. Unoccupancy of the QB site by plastoquinone partially protected RCII against the light-induced destabilization of Q-B. The extent of charge separation (P+680Q-A) was not affected. However, the slow phase (microsecond) of P+680 dark reduction increased, and the amplitude of signal II was reduced by 20-30%, indicating that in a fraction of RCII, electron donation from Z to P+680 was impaired without losing primary photochemistry. This modification correlates with the irreversible change in D1 protein resulting in the formation of a trypsin-resistant fragment of 16 kDa detected in D1 isolated from light-exposed cells. The change in the Q-B stability could allow charge equilibration with QA and thus explain the rise in the intrinsic fluorescence level and reduction of electron flow to plastoquinone. The change in the lifetime of P+680 can account for further reduction in electron flow (photo-inhibition). The irreversible light-dependent modification of D1 may serve as the signal for its degradation and replacement by a newly synthesized molecule (turnover).     

Trypsin-sensitive photosynthetic activities in chloroplast membranes from Chlamydomonas reinhardi, y-1.

Location of electron transport chain components in chloroplast membranes of chlamydomonas reinhardi, y-1 was investigated by use of proteolytic digestion with soluble or insolubilized trypsin. Digestion of intact membrane vesicles with soluble trypsin inactivates the water-splitting system, the 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition site of Photosystem II, the electron transport between the two photosystems as well as the ferredoxin NADP reductase. Reduction of NADP with artificial electron donors for Photosystem I could be restored, however, by addition of purified reductase to trypsin-digested membranes. Electron transfer activities of Photosystems I and II reaction centers were resistant to trypsin digestion either from outside or from within the thylakoids when active trypsin was trapped inside the membrane vesicles by sonication and digestion carried out in the presence of trypsin inhibitor added from outside. In the latter case, the water-splitting system was also found to be resistant to digestion. Polyacrylamide-bound insolubilized trypsin inactivated only the ferredoxin NADP reductase. Photosynthetically active membranes obtained at different stages of development showed a basically similar behavior toward trypsin.     

Differentiation and development of bundle sheath and mesophyll thylakoids in maize. Thylakoid polypeptide composition, phosphorylation, and organization of photosystem II

Photosynthetic electron flow, polypeptide pattern, presence of chlorophyll-protein complexes, and phosphorylation of thylakoid polypeptides have been investigated in differentiated mesophyll (M) and bundle sheath (B) thylakoids of the C4 plant Zea mays. The polypeptide pattern of M thylakoids and their photosynthetic electron flow are comparable to those of other green plants. B thylakoids exhibit only photosystem I (PSI) activity, contain only traces of the PSII light harvesting (LHCII) polypeptide, do not bind [3H] diuron, and lack polypeptides of the water-oxidation complex of PSII and the herbicide binding 32-kDa polypeptide, as detected by specific antibodies. However, B thylakoids possess a partially active PSII reaction center, as demonstrated by light-dependent reduction of silicomolybdate with 1,5-diphenylcarbazide (DPC) as an electron donor, and the presence of the PSII reaction center polypeptides of 44-47 kDa. Only one chlorophyll a-protein complex, corresponding to the PSI reaction center-core antenna, was detectable in B thylakoids, as opposed to chlorophyll a and chlorophyll a,b-protein complexes present in M thylakoids. The light-dependent, membrane-bound kinase activity present in M thylakoids could not be detected in B thylakoids which, nevertheless, contain a protein kinase able to phosphorylate casein. A total of 19 differences between the electrophoretic pattern of B and M thylakoid polypeptides were observed. The mRNA coding for the LHCII polypeptide is primarily, if not exclusively, localized in M cells. The development of PSII complex precedes that of PSI during the differentiation of B and M chloroplasts in expanding leaves of light-grown plants and during the greening of dark-grown etiolated seedlings. The differentiation of the maize leaf into cells programmed to form B or M chloroplasts does not require light. In light-grown plants, the differentiation of B and M thylakoids occurred progressively from the base of the leaf and was completed at 4-5 cm from the leaf base.     

<Emphasis Type="Italic">Physcomitrella patens</Emphasis> and <Emphasis Type="Italic">Ceratodon purpureus</Emphasis>, mosses as model organisms in photosynthesis studies

With the discovery of targeted gene replacement, moss biology has been rapidly advancing over the last 10 years. This study demonstrates the usefulness of moss as a model organism for plant photosynthesis research. The two mosses examined in this study, Physcomitrella patens and Ceratodon purpureus, are easily cultured through vegetative propagation. Growth tests were conducted to determine carbon sources suitable for maintaining heterotrophic growth while photosynthesis was blocked. Photosynthetic parameters examined in these plants indicated that the photosynthetic activity of Ceratodon and Physcomitrella is more similar to vascular plants than cyanobacteria or green algae. Ceratodon plants grown heterotrophically appeared etiolated in that the plants were taller and plastids did not differentiate thylakoid membranes. After returning to the light, the plants developed green, photosynthetically active chloroplasts. Furthermore, UV-induced mutagenesis was used to show that photosynthesis-deficient mutant Ceratodon plants could be obtained. After screening approximately 1000 plants, we obtained a number of mutants, which could be arranged into the following categories: high fluorescence, low fluorescence, fast and slow fluorescence quenching, and fast and slow greening. Our results indicate that in vivo biophysical analysis of photosynthetic activity in the mosses can be carried out which makes both mosses useful for photosynthesis studies, and Ceratodon best sustains perturbations in photosynthetic activity.     

Identification of methylated sequences in genomic DNA of adult Drosophila melanogaster

The genome of Drosophila melanogaster contains methylated cytosines. Recent studies indicate that DNA methylation in the fruit fly depends on one DNA methyltransferase, dDNMT2. No obvious phenotype is associated with the downregulation of this DNA methyltransferase. Thus, identifying the target sequences methylated by dDNMT2 may constitute the first step towards understanding the biological functions of this enzyme. We used anti-5-methylcytosine antibodies as affinity column to identify the methylated sequences in the genome of adult flies. Our analysis demonstrates that components of retrotransposons and repetitive DNA sequences are putative substrates for dDNMT2. The methylation status of DNA encoding Gag, a protein involved in delivering the transposition template to its DNA target, was confirmed by sodium bisulfite sequencing.