A Periplasmic LolA Derivative with a Lethal Disulfide Bond Activates the Cpx Stress Response System

LolA accommodates the acyl chains of lipoproteins in its hydrophobic cavity and shuttles between the inner and outer membranes through the hydrophilic periplasm to place lipoproteins in the outer membrane. The LolA(I93C/F140C) derivative, in which Cys replaces Ile at position 93 and Phe at position 140, strongly inhibited growth in the absence of a reducing agent because of the lethal intramolecular disulfide bond between the two Cys residues. Expression of I93C/F140C was found to activate the Cpx two-component system, which responds to cell envelope stress. The inhibition of growth by I93C/F140C was partly suppressed by overproduction of LolCDE, which is an ATP-binding cassette transporter and mediates the transfer of lipoproteins from the inner membrane to LolA. A substantial portion of the oxidized form, but not the reduced one, of I93C/F140C expressed on LolCDE overproduction was recovered in the membrane fraction, whereas wild-type LolA was localized in the periplasm even when LolCDE was overproduced. Moreover, LolCDE overproduction stabilized I93C/F140C and therefore caused an increase in its level. Taken together, these results indicate that oxidized I93C/F140C stably binds to LolCDE, which causes strong envelope stress.There are more than 90 different species of lipoproteins in the Escherichia coli envelope, most of which are localized on the periplasmic side of the outer membrane (29, 31) They each have an N-terminal cysteine covalently modified with three acyl chains, and are anchored to membranes via these lipid tails (25). Although some of these proteins have been shown to be involved in important cellular processes, such as biogenesis of the outer membrane (1, 14, 24, 35), drug transport (11), and signal transduction (7), the functions of the majority of them remain unknown. The Lol system, composed of five Lol proteins, is required for the sorting and targeting of outer membrane-specific lipoproteins (30).Lipoprotein precursors are sequentially processed to their mature forms on the periplasmic side of the inner membrane after their translocation across the inner membrane by Sec translocon (21). Those destined for the outer membrane then each form a complex with LolCDE (36), a member of ATP-binding cassette (ABC) transporter family, in the inner membrane. LolA, a periplasmic lipoprotein-specific carrier, receives lipoproteins from LolCDE in an ATP hydrolysis-dependent manner and forms a water-soluble complex with a lipoprotein (13). The complex then traverses the periplasmic space from the inner to the outer membrane, where lipoproteins are transferred from LolA to a lipoprotein receptor, LolB (14), in a mouth-to-mouth manner (20). Finally, lipoproteins are anchored to the outer membrane through the action of LolB (32).LolA is composed of 11 antiparallel β-sheets and 3 α-helices, which form an incomplete β-barrel structure with a lid covering the barrel (28). The cavity formed inside the barrel is hydrophobic and is considered to be the binding site for the acyl chains of lipoproteins. To elucidate the role of the opening and closing of the hydrophobic cavity in lipoprotein transfer reactions, the LolA(I93C/F140C) mutant, in which cysteine replaces Ile93 in the α2 helix and Phe140 in the β10 strand, was previously constructed (34). In I93C/F140C expressed in the periplasm, an intramolecular disulfide bond was formed between the two cysteine residues. This oxidized form of I93C/F140C was fixed in the closed conformation and was unable to release lipoproteins from the inner membrane, suggesting that opening of the cavity is crucial for the LolA function (34). Biochemical studies subsequently showed that the LolA cavity indeed undergoes opening and closing upon the binding and release of lipoproteins, respectively (19). Moreover, it was found that only the closed form of LolA is active in the lipoprotein release reaction.I93C/F140C exhibited the strongest growth inhibition among the LolA mutants so far isolated, although it was fully active in the presence of a reducing agent (34). We show here that oxidized I93C/F140C strongly activates the Cpx two-component system (23) that responds to cell envelope stress, whereas overproduction of LolCDE partly suppresses the toxic effect of I93C/F140C.     

Identification and Characterization of an Assembly Intermediate Subcomplex of Photosystem I in the Green Alga Chlamydomonas reinhardtii

Photosystem I (PSI) is a multiprotein complex consisting of the PSI core and peripheral light-harvesting complex I (LHCI) that together form the PSI-LHCI supercomplex in algae and higher plants. The supercomplex is synthesized in steps during which 12–15 core and 4–9 LHCI subunits are assembled. Here we report the isolation of a PSI subcomplex that separated on a sucrose density gradient from the thylakoid membranes isolated from logarithmic growth phase cells of the green alga Chlamydomonas reinhardtii. Pulse-chase labeling of total cellular proteins revealed that the subcomplex was synthesized de novo within 1 min and was converted to the mature PSI-LHCI during the 2-h chase period, indicating that the subcomplex was an assembly intermediate. The subcomplex was functional; it photo-oxidized P700 and demonstrated electron transfer activity. The subcomplex lacked PsaK and PsaG, however, and it bound PsaF and PsaJ weakly and was not associated with LHCI. It seemed likely that LHCI had been integrated into the subcomplex unstably and was dissociated during solubilization and/or fractionation. We, thus, infer that PsaK and PsaG stabilize the association between PSI core and LHCI complexes and that PsaK and PsaG bind to the PSI core complex after the integration of LHCI in one of the last steps of PSI complex assembly.     

Analysis of DNA and Zinc finger interactions using mechanical force spectroscopy

The unbinding force of Zif268-DNA complex has been studied by atomic force microscopy (AFM). DNA and Zif268 were covalently immobilized on the surfaces of an AFM tip and glass substrate, respectively. Confocal microscopy was used to confirm the successful immobilization of DNA. Because of the complexity of the protein-DNA interaction, parallel experiments were designed to discriminate specific interactions. For such experiments, a typical unbinding force of a single Zif268-DNA complex (approx 550 pN at 40 nN/s force loading rate) was evaluated.     

Production of IL-13 in spleen cells by IL-18 and IL-12 through generation of NK-like cells

Treatment of Nylon wool-passed cells (NWC) prepared from the spleen of C57BL/6 mice with IL-18 and IL-12, but not with IL-18 alone, resulted in induction of IFN-gamma, a Th1 cytokine, and GM-CSF at 24 h, and IL-13, a Th2 cytokine at 72 h. The induction of IL-13 was suppressed by anti-GM-CSF antibody, indicating involvement of GM-CSF in IL-13 production. When NWC incubated with IL-18 and IL-12 for 72 h ("primary treatment") were treated again with the same cytokines ("secondary treatment"), IL-13 was induced much more quickly than observed in the primary treatment. Flow cytometric analysis of NWC after the primary treatment showed marked increases in the CD4(-)CD8(-) non-T cell population bearing CD25(+), CD45RB(super high) and CD122(+). These cells were positive for CD49b but negative for NK1.1, indicating that they were not typical but NK-like cells. The NK-like cells produced IL-13 in response to the treatment with IL-18 alone, indicating that the generation of these cells in the primary treatment likely accounts for the quick production of IL-13 in the secondary treatment. These results show that IL-18 and IL-12 generates the NK-like cells in NWC by a process mediated by GM-CSF that are ready for producing IL-13.     

Effect of Japanese Paramecium bursaria extract on photosynthetic carbon fixation of symbiotic algae

To investigate the relationship between the Japanese Paramecium bursaria host and its symbiont, we studied the effect of a host cell-free extract on carbon fixation and photosynthate release of the symbiont. The host extract enhanced symbiotic algal carbon fixation about 3-fold at an increased concentration; however, release of photosynthate hardly changed. Since the enhancing effect was not affected by elimination of carbon dioxide from the host extract, the existence of a host factor that stimulates algal carbon fixation was made clear. The host factor is a heat-stable, low molecular weight substance. In relation to the pH dependence, the extract improved carbon fixation at acidic and neutral pH and showed almost no effect at pH 9.0. Therefore, the stimulation of carbon fixation by the host factor is unlikely to be caused by intracellular pH change. The extract also improved carbon fixation of several Chlorella species, symbiotic and free-living, and apparently exhibited no species specificity. Therefore, the host seems to regulate the photosynthesis of the symbiont via a specific compound.     

Dynamics of Golgi matrix proteins after the blockage of ER to Golgi transport

When the ER to Golgi transport is blocked by a GTP-restricted mutant of Sar1p (H79G) in NRK-52E cells, most Golgi resident proteins are transported back into the ER. In contrast, the cis-Golgi matrix proteins GM130 and GRASP65 are retained in punctate cytoplasmic structures, namely Golgi remnants. Significant amounts of the medial-Golgi matrix proteins golgin-45, GRASP55 and giantin are retained in the Golgi remnants, but a fraction of these proteins relocates to the ER. Golgin-97, a candidate trans-Golgi network matrix protein, is retained in Golgi remnant-like structures, but mostly separated from GM130 and GRASP65. Interestingly, most Sec13p, a COPII component, congregates into larger cytoplasmic clusters soon after the microinjection of Sar1p(H79G), and these move to accumulate around the Golgi apparatus. Sec13p clusters remain associated with Golgi remnants after prolonged incubation. Electron microscopic analysis revealed that Golgi remnants are clusters of larger vesicles with smaller vesicles, many of which are coated. GM130 is mainly associated with larger vesicles and Sec13p with smaller coated vesicles. The Sec13p clusters disperse when p115 binding to the Golgi apparatus is inhibited. These results suggest that cis-Golgi matrix proteins resist retrograde transport flow and stay as true residents in Golgi remnants after the inhibition of ER to Golgi transport.     

Disruption of the Plasmodium falciparum 2-Cys peroxiredoxin gene renders parasites hypersensitive to reactive oxygen and nitrogen species

In parasitism, Plasmodium falciparum is exposed to toxic reactive oxygen species and reactive nitrogen species (RNS). Peroxiredoxins (Prx) are ubiquitously distributed antioxidant enzymes. In bacteria and yeast, Prx have also been implicated in detoxifying RNS. Here, we used a gene targeting strategy to investigate the physiological role of 2-Cys Prx of P. falciparum, PfTPx-1, in living parasite cells. The PfTPx-1-null parasite line was more sensitive to paraquat (a superoxide donor) and sodium nitroprusside (a nitric oxide donor), than wildtype. These findings suggest that PfTPx-1 protects the parasite cells from oxidative and nitrosative stresses.     

New approach for the establishment of an hepatocyte cell line derived from rat early embryonic stem cells

A cell line with the characteristics of hepatocytes was established from rat early embryonic stem cells (REES). This cell line was established using a new novel method of Ishiwata et al. from two cell embryos taken from the spontaneous dwarf rat (SDR). The hepatocyte cell line (REES-hep) was instituted from dark red colored tissue in embryos during embryogenesis using REES cell line cultured in the presence of embryotrophic factors. These cell lines were cultured with DMEM/F12 medium supplemented 10% FBS and 1 ng/ml of LIF. They were found to maintain their diploid state, were characterized with 42 normal chromosomes and proliferated to confluence; contact inhibition was also present. These cells produced albumin when cultured using a collagen sponge gel system and reconstructed in a funicular form resembling the cell cords of liver. The cells also produced albumin and bilirubin when transplanted into the spleen of SDR Reconstruction of a REES-hep cell line from early embryonic stem cells should help in treating hepatic insufficient patients. It will be valuable for further research, as an introduction to cell transplantation and application for use in a bio-hybrid typed liver apparatus.     

Improved silencing vector co-expressing GFP and small hairpin RNA

Small interfering RNA (siRNA) is a powerful tool for the specific silencing of gene expression. We developed an improved vector, pG-SUPER, that co-expresses green fluorescent protein (GFP) and small hairpin RNA simultaneously to facilitate analysis of silencing at the level of individual cells. As a test system, we analyzed lamin A/C knockdown in HeLa cells. The GFP signal was a reliable reporter (93%-98%) of strong knockdown (approximately 90%) over a wide range of GFP intensities. The GFP reporter made possible the application of fluorescent-activated cell sorting (FACS) to purify the knockdown cell population. Such populations facilitated Western blotting analysis to determine depletion of the target protein. pG-SUPER was also applied to evaluate gene replacement by exogenous genes rendered refractory to siRNA by introducing silent mutations. Recovery of lamin A was linearly correlated to the expression level of the rescue gene. pG-SUPER will expand plasmid-based siRNA applications through the easy and reliable detection of knockdown and rescued cells.