A method was developed to quantify the fraction of photosystem I (PSI) centers that operate according to the cyclic or linear mode, respectively. P(700) and plastocyanin oxidation were analyzed under a weak far-red excitation (approximately eight photons per s(-1) per PSI) that induces P(700) oxidation in approximately 20 s and approximately 3 s in dark-adapted and preilluminated leaves, respectively. This finding implies that, in dark-adapted leaves, most of the electrons formed on the stromal side of PSI are transferred back to PSI through an efficient cyclic chain, whereas in preilluminated leaves, electrons are transferred to NADP and then to the Benson-Calvin cycle. Preillumination thus induces a transition from the cyclic to the linear mode. A reverse transition occurs in the dark in a time that increases with the duration and intensity of preillumination. After a approximately 10-min illumination under strong light that activates the Benson-Calvin cycle, the transition from the linear to the cyclic mode is completed in >1 h (t(1/2) approximately 30 min). The fraction of PSI involved in the cyclic process in dark-adapted leaves can be close to 100%. An apparent equilibrium constant of approximately 4 between P(700) and plastocyanin was measured during the course of the far-red illumination. This value is much lower than that computed from the midpoint redox potential of the two carriers (approximately 30). These results are interpreted assuming that chloroplasts include isolated compartments defined on the basis of the structural organization of the photosynthetic chain proposed by Albertsson [Albertsson, P. A. (2001) Trends Plant Sci. 6, 349-354].

The kinetics of reoxidation of the primary acceptor Qa has been followed by measuring the changes in the fluorescence yield induced by a series of saturating flashes in intact cells of Rhodobacter sphaeroides in anaerobic conditions. At 0 °C, about half of Qa− is reoxidized in about 200 ms while reoxidation of the remaining fraction is completed in several seconds to minutes. The fast phase is associated with the transfer of ubiquinone formed at site Qo of the cytochrome bc1 complex while the slowest phase is associated with the diffusion of ubiquinone present in the membrane prior to the flash excitation. The biphasic kinetics of Qa− oxidation is interpreted assuming that the electron chain is organized in supercomplexes that associate two RCs and one cyt bc1 complex, which allows a fast transfer of quinone formed at the level of cyt bc1 complex to the RCs. In agreement with this model, the fast phase of Qa− reoxidation is inhibited by myxothiazol, a specific inhibitor of cyt bc1. The PufX-deleted mutant displays only the slowest phase of Qa− oxidation; it is interpreted by the lack of supramolecular organization of the photosynthetic chain that leads to a larger average distance between cyt bc1 and RCs.

The rate of cyclic electron flow measured in dark-adapted leaves under aerobic conditions submitted to a saturating illumination has been performed by the analysis of the transmembrane potential changes induced by a light to dark transfer. Using a new highly sensitive spectrophotometric technique, a rate of the cyclic flow of ∼130 s−1 has been measured in the presence or absence of 3-(3,4-dichloro-phenyl)-1,1-dimethylurea (DCMU). This value is ∼1.5 times larger than that previously reported [Proc. Natl. Acad. Sci. U. S. A. 99 (2001) 10209]. We have characterized in the presence or absence of DCMU charge recombination process (t1/2∼60 μs) that involves P700+ and very likely the reduced form of the iron sulfur acceptor FX. This led to conclude that, under saturating illumination, the PSI centers involved in the cyclic pathway have most of the iron sulfur acceptors FA and FB reduced. In the proposed mechanism, electrons are transferred from a ferredoxin bound to a site localized on the stromal side of the cytochrome b6f complex to the Qi site. Two possible models of the organization of the membrane complexes are discussed, in which the cyclic and linear electron transfer chains are isolated one from the other.

In Arabidopsis, the nuclear genes PetC and AtpD code for the Rieske protein of the cytochrome b(6)/f (cyt b(6)/f) complex and the delta-subunit of the chloroplast ATP synthase (cpATPase), respectively. Knock-out alleles for each of these loci have been identified. Greenhouse-grown petc-2 and atpd-1 mutants are seedling lethal, whereas heterotrophically propagated plants display a high-chlorophyll (Chl)-fluorescence phenotype, indicating that the products of PetC and AtpD are essential for photosynthesis. Additional effects of the mutations in axenic culture include altered leaf coloration and increased photosensitivity. Lack of the Rieske protein affects the stability of cyt b(6)/f and influences the level of other thylakoid proteins, particularly those of photosystem II. In petc-2, linear electron flow is blocked, leading to an altered redox state of both the primary quinone acceptor Q(A) in photosystem II and the reaction center Chl P700 in photosystem I. Absence of cpATPase-delta destabilizes the entire cpATPase complex, whereas residual accumulation of cyt b(6)/f and of the photosystems still allows linear electron flow. In atpd-1, the increase in non-photochemical quenching of Chl fluorescence and a higher de-epoxidation state of xanthophyll cycle pigments under low light is compatible with a slower dissipation of the transthylakoid proton gradient. Further and clear differences between the two mutations are evident when mRNA expression profiles of nucleus-encoded chloroplast proteins are considered, suggesting that the physiological states conditioned by the two mutations trigger different modes of plastid signaling and nuclear response.

The turnover of linear and cyclic electron flows has been determined in fragments of dark-adapted spinach leaf by measuring the kinetics of fluorescence yield and of the transmembrane electrical potential changes under saturating illumination. When Photosystem (PS) II is inhibited, a cyclic electron flow around PSI operates transiently at a rate close to the maximum turnover of photosynthesis. When PSII is active, the cyclic flow operates with a similar rate during the first seconds of illumination. The high efficiency of the cyclic pathway implies that the cyclic and the linear transfer chains are structurally isolated one from the other. We propose that the cyclic pathway operates within a supercomplex including one PSI, one cytochrome bf complex, one plastocyanin, and one ferredoxin. The cyclic process induces the synthesis of ATP needed for the activation of the Benson–Calvin cycle. A fraction of PSI (∼50%), not included in the supercomplexes, participates in the linear pathway. The illumination would induce a dissociation of the supercomplexes that progressively increases the fraction of PSI involved in the linear pathway.

The kinetics and amplitude of the membrane potential changes associated with electron and proton transfers within the cytochrome b(6)/f (cyt b/f) complex (phase b) are measured in vivo in Chlamydomonas reinhardtii under anaerobic conditions. Upon saturating flash excitation, fast components in the membrane potential decay superimposed on phase b lead to an underestimation of the amplitude of this phase. In the FUD50 mutant strain, which lacks the ATP synthase, the decay of the membrane potential is slowed down compared to the wild type, and the kinetics and amplitude of phase b may be accurately determined. This amplitude corresponds to the transfer of at least 1.5 charges across the membrane per positive charge transferred to photosystem I, whatever the flash energy. This value largely exceeds that predicted by a Q-cycle process. Similar conclusions are reached using the wild type strain in the presence of 9 microM dicyclohexylcarbodiimide, which specifically inhibits the ATP synthase. It is concluded that a proton pumping process is operating in parallel with the Q-cycle, with a yield of approximately 0.5 proton pumped by cyt b/f complex turnover, irrespective of the flash energy.

All photosynthetic reaction centers share a common structural theme. Two related, integral membrane polypeptides sequester electron transfer cofactors into two quasi-symmetrical branches, each of which incorporates a quinone. In type II reaction centers [photosystem (PS) II and proteobacterial reaction centers], electron transfer proceeds down only one of the branches, and the mobile quinone on the other branch is used as a terminal acceptor. PS I uses iron-sulfur clusters as terminal acceptors, and the quinone serves only as an intermediary in electron transfer. Much effort has been devoted to understanding the unidirectionality of electron transport in type II reaction centers, and it was widely thought that PS I would share this feature. We have tested this idea by examining in vivo kinetics of electron transfer from the quinone in mutant PS I reaction centers. This transfer is associated with two kinetic components, and we show that mutation of a residue near the quinone in one branch specifically affects the faster component, while the corresponding mutation in the other branch specifically affects the slower component. We conclude that both electron transfer branches in PS I are active.

Electron transfer within PS I reaction centers has been analyzed in vivo in a mutant of Chlorella sorokiniana which lacks most of the PS II and of the peripheric antennae, using a new spectrophotometric technique with a time resolution of approximately 5 ns. Absorption changes associated with the oxidation of semiphylloquinone (acceptor A(1)(-)) have been characterized in the 371-545 nm spectral range. The oxidation of A(1)(-) and the reduction of an iron-sulfur cluster (F(X), F(A)F(B)) is monitored by an absorption decrease at 377 nm (semiphylloquinone absorption band) and by the decrease of two positive absorption bands around 480 and 515 nm, respectively, very likely associated with a local electrochromic shift induced by A(1)(-) on a carotenoid molecule localized in its vicinity. A(1)(-) undergoes a two-phase oxidation of about equal amplitude with half-times of approximately 18 and approximately 160 ns, respectively. Two hypotheses are proposed to interpret these data: (1) Photosystem I reaction centers are present under two conformational states which differ by the reoxidation rate of A(1)(-). (2) The two phylloquinones corresponding to the two branches of the PS I heterodimer are involved in the electron transfer. The similar amplitude of the two phases implies that the rates of electron transfer from P700 to each of the phylloquinones are about equal. The two different rate constants measured for A(1)(-) oxidation suggests some asymmetry in the relative position of the two phylloquinones with respect to F(X).

Engrailed homeoprotein, a transcription factor involved in midbrain/hindbrain patterning, primarily localizes to the cell nucleus. However, significant amounts of the protein are also found in the cell cytoplasm or associated with membrane microdomains enriched in cholesterol and glycosphingoglycolipids (Joliot, A., Trembleau, A., Raposo, G., Calvet, S., Volovitch, M. and Prochiantz, A. (1997) Development 124, 1865-1875). This non-nuclear localization, observed in vitro and in vivo, led us to investigate the possibility that Engrailed be transferred between nuclear and non-nuclear compartments. Monkey COS-7 cells expressing chick Engrailed-2 (cEN2) were fused with 3T3 mouse fibroblasts and the passage of cEN2 from COS-7 to 3T3 nuclei was followed in the interspecies heterokaryons. We find that, 10 minutes following cell fusion, cEN2 is detected in the 3T3 nuclei of 80% of the heterokaryons demonstrating rapid cEN2 nuclear export. Export from donor nuclei can be saturated and is strongly reduced after deletion of a 11 amino acid-long (&Dgr;)1 sequence present within a slightly larger domain that extends between helices 2 and 3 of the homeodomain and shows strong similarities with leucine-rich nuclear export signals (NES). This putative NES, when fused with a nuclear reporter protein, allows its nuclear export, demonstrating that it is not only necessary but also sufficient for nuclear export and can therefore be considered as a true nuclear export sequence. In an earlier report (Joliot, A., Maizel, A., Rosenberg, D., Trembleau, A., Dupas, S., Volovitch, M. and Prochiantz, A. (1998) Current Biology 8, 856-863), we demonstrated that the (&Dgr;)1 sequence is necessary for the access of cEN2 to the lumen of a membrane compartment and for its intercellular transfer. The present study thus strongly suggests that the regulation of Engrailed nuclear export could play a role not only in Engrailed transcriptional activity but also in its ability to gain access to a secretory compartment.

The effect of N,N'-dicyclohexylcarbodiimide (DCCD) on electron and proton transfers within the cytochrome (cyt) bf complex has been analyzed in living cells of the green algae Chlorella sorokiniana under anaerobic conditions. DCCD induces a partial decoupling of the protomotive Q-cycle, in agreement with the conclusions of Wang and Beattie (1991) Arch. Biochem. Biophys. 291, 363-370. In the presence of 20 microM DCCD, we observe the development of a lag phase in the kinetics of the slow electrogenic phase associated with electron and proton transfers within the cyt bf complex. In the same conditions, the initial rate of cyt b and cyt f reduction is decreased by about 30%. We propose that in the absence of DCCD, a transmembrane movement of proton is coupled to the oxidation of plastoquinol at site Qo. In the presence of 20 microM DCCD, this redox-coupled proton pump is inhibited, and the kinetics of phase b and cyt b reduction become close to that predicted on the basis of a pure Q-cycle process. In agreement with this hypothesis, we observe that upon a weak-flash excitation, two charges are translocated through the membrane in addition to the charge translocated at the level of photosystem I. Part of this large electrogenic phase could be associated with the translocation of a proton from the stroma to the lumen. A tentative mechanism is discussed that remains in the frame of the Q-cycle but accounts for an additional proton-pumping process or for the partial decoupling observed in the presence of DCCD, as well.

The cytochrome (cyt) bf complex catalyses the oxidation of plastoquinone and the reduction of plastocyanine. Electron transfer within the cyt bf complex induces a proton-pumping and the formation of a membrane potential.

Background: Engrailed-land Engrailed-2 are homeoproteins - transcription factors implicated in the morphogenesis of discrete structures. Engrailed proteins have a role in patterning the midbrain-hindbrain region and are expressed in the nuclei of rat embryo midbrain-hindbrain cells. We have previously found that both endogenous and exogenously expressed Engrailed proteins also associate with membrane regions implicated in signal transduction and secretion. Within total membrane fractions, a small proportion of Engrailed - about 5% - is protected against proteinase K proteolysis, suggesting that Engrailed has access to a luminal compartment. Together with our finding that homeodomains and homeoproteins can be internalized by live cells, these observations suggest that Engrailed might act as a polypeptidic messenger. In order to investigate this possibility, we looked to see if Engrailed could be secreted.Results: Engrailed expressed in COS cells can be recovered in abutting primary neurons and this is dependent on a short sequence in its homeodomain distinct from 'classical' secretion signals. This sequence, which overlaps with the sequence necessary for Engrailed internalization and which is highly conserved among homeoproteins, is the first example of an 'unconventional' sequence necessary for secretion. Less than 5% of total intracellular Engrailed is secreted and there is a correlation between secretion and access to the membrane compartment where the protein is protected against proteinase K,Conclusions: Our results lend weight to the proposal that Engrailed, and possibly other homeoproteins, might act as intercellular polypeptidic messengers.

Biology of the CellVolume 90, Issue 1 p. 100-101 Going in without endocytosis and out without signal sequence: Engrailed can do it! A. Joliot, A. Joliot Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this authorA. Trembleau, A. Trembleau Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this authorD. Derossi, D. Derossi Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this authorG. Chassaing, G. Chassaing Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this authorA. Maizel, A. Maizel Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this authorM. Volovitch, M. Volovitch Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this authorAlain Prochiantz, Alain Prochiantz Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this author A. Joliot, A. Joliot Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this authorA. Trembleau, A. Trembleau Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this authorD. Derossi, D. Derossi Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this authorG. Chassaing, G. Chassaing Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this authorA. Maizel, A. Maizel Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this authorM. Volovitch, M. Volovitch Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this authorAlain Prochiantz, Alain Prochiantz Développement et Evolution du Système Nerveux, Section Biologie, ENS, 46 rue d'Ulm, 75230 Paris cedex 05, FranceSearch for more papers by this author First published: 01 February 2012 https://doi.org/10.1016/S0248-4900(98)80242-6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume90, Issue1January 1998Pages 100-101 RelatedInformation

The three phases of the flash-induced membrane potential rise were measured in Rhodobacter sphaeroides, at temperatures between 16°C and −30°C, in the liquid or the frozen state. We show that phase II, which is myxothiazol-insensitive, includes phase IIa and phase IIb, completed at −8°C in about 2 ms and 10 ms, respectively. Phase IIa is very likely associated with the protonation of the doubly-reduced quinone acceptor QB= (Drachev, L.A., Mamedov, A., Mulkidjanian, A.Y., Semenov, A.Y., Shinkarev, V.P. and Verkhovsky, M.J. (1988) FEBS Lett. 233, 315–318); phase IIb is associated with the oxidation of cytochrome c2 (Jackson, J.B. and Dutton, P.L., 1973, Biochim. Biophys. Acta 325, 102–113). Freezing the sample does not modify the kinetics of phase IIa but slows down phase IIb by a factor of 2. The amplitude of phase III, which is exclusively related to electron and proton transfer within the cytochrome b/c1 complex, is temperature-independent between room temperature and −16°C. In the frozen state, the rate of phase III is mainly limited by the electron transfer from cytochrome c2 to the reaction centers and not by the movement of cytochrome c2 between the two membrane complexes. These results are interpreted assuming that one cytochrome c2 is trapped in a supercomplex formed by the association of two reaction centers and one cytochrome b/c1 complex.

We report here that the homeoproteins Engrailed-1 and Engrailed-2 are present in specific non-nuclear subcellular compartments. Using electron microscopy, we observed that chick-Engrailed-2 expressed in COS-7 cells associates with membrane fractions that are characterized as caveolae. This characterization is based on morphological, biochemical and immunological criteria such as, in particular, the absence of clathrin coat and the presence of caveolin and cholera toxin-binding sites. These data are fully confirmed by subcellular fractionation experiments, which demonstrate that transfected chick-Engrailed-2 is present in low density membrane fractions that are resistant to Triton X-100, enriched in caveolin and solubilized by the addition of a cholesterol-binding detergent, a set of properties highly characteristic of caveolae. The association of Engrailed-2 with specific membrane fractions observed after transfection in COS-7 cells is also observed for endogenous Engrailed-1 and Engrailed-2 expressed at late embryonic stages in the cerebellum and posterior mesencephalon of the rodent. Indeed, the two proteins are present in membrane fractions that bear all the characteristics of microdomains or caveolae-like domains, i.e. Triton X-100 resistance, saponin solubilization, low density on sucrose gradients, enrichment in glycosphingolipid GM1, absence of transmembrane Neural Cell Adhesion Molecule, presence of the glypiated (GPI-anchored) glycoprotein F3/F11 and of the acylated growth-associated protein GAP-43. Finally we demonstrate that part of the membrane-associated Engrailed, either expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have been permeabilized with detergent. This study suggests that, in addition to their well-known presence in the nucleus, Engrailed proteins are also associated with caveolae-like vesicles that are primarily transported anterogradely into the axon, and that they can get access to a compartment compatible with secretion.