Multiple pathways for protein transport into or across the thylakoid membrane

Many thylakoid proteins are cytosolically synthesized and have to cross the two chloroplast envelope membranes as well as the thylakoid membrane en route to their functional locations. In order to investigate the localization pathways of these proteins, we over-expressed precursor proteins in Escherichia coli and used them in competition studies. Competition was conducted for import into the chloroplast and for transport into or across isolated thylakoids. We also developed a novel in organello method whereby competition for thylakoid transport occurred within intact chloroplasts. Import of all precursors into chloroplasts was similarly inhibited by saturating concentrations of the precursor to the OE23 protein. In contrast, competition for thylakoid transport revealed three distinct precursor specificity groups. Lumen-resident proteins OE23 and OE17 constitute one group, lumenal proteins plastocyanin and OE33 a second, and the membrane protein LHCP a third. The specificity determined by competition correlates with previously determined protein-specific energy requirements for thylakoid transport. Taken together, these results suggest that thylakoid precursor proteins are imported into chloroplasts on a common import apparatus, whereupon they enter one of several precursor-specific thylakoid transport pathways.     

A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen

We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single-copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin-like C-terminal segment of 20 kDa, a predicted N-terminal leucine zipper and a potential phosphatase-binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non-appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl-prolyl cis-trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light-mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated.     

Sec/SRP-independent insertion of two thylakoid membrane proteins bearing cleavable signal peptides

Two imported thylakoid membrane proteins, PSII-X and PSII-W, are synthesised with cleavable N-terminal signal peptides that closely resemble those of Sec-dependent lumenal proteins. In this report we have reconstituted the insertion of pre-PSII-X and pre-PSII-W into isolated thylakoids. We show that insertion does not require either nucleoside triphosphates or stromal extracts, both of which are required for Sec- and signal recognition particle (SRP)-dependent targeting mechanisms. Insertion is furthermore unaffected by protease treatments that destroy the known protein translocation apparatus in the thylakoid membrane. We conclude that these membrane proteins are inserted by an unusual Sec/SRP-independent mechanism that probably resembles that used by CFoII, and we discuss possible parallels with the biogenesis of phage M13 procoat.     

Periodic variations in the ratio of free to thylakoid-bound chloroplast ribosomes during the cell cycle of Chlamydomonas reinhardtii

The ratio of free to thylakoid-bound chloroplast ribosomes in Chlamydomonas reinhardtii undergoes periodic changes during the synchronous light-dark cycle. In the light, when there is an increase in the chlorophyll content and synthesis of thylakoid membrane proteins, about 20-30% of the chloroplast ribosomes are bound to the thylakoid membranes. On the other hand, only a few or no bound ribosomes are present in the dark when there is no increase in the chlorophyll content. The ribosome-membrane interaction depends not only on the developmental stage of the cell but also on light. Thus, bound ribosomes were converted to the free variety after cultures at 4 h in the light had been transferred to the dark for 10 min. Conversely, a larger number of chloroplast ribosomes became attached to the membranes after cultures at 4 h in the dark had been illuminated for 10 min. Under normal conditions, when there was slow cooling of the cultures during cell harvesting, chloroplast polysomal runoff occurred in vivo leading to low levels of thylakoid-bound ribosomes. This polysomal runoff could be arrested by either rapid cooling of the cells or the addition of chloramphenicol or erythromycin. Each of these treatments prevented polypeptide chain elongation on chloroplast ribosomes and thus allowed the polyosomes to remain bound to the thylakoids. Addition of lincomycin, an inhibitor of chain initiation on 70S ribosomes, inhibited the assembly of polysome-thylakoid membrane complex in the light. These results support a model in which initiation of mRNA translation begins in the chloroplast stroma, and the polysome subsequently becomes attached to the thylakoid membrane. Upon natural chain termination, the chloroplast ribosomes are released from the membrane into the stroma.     

The thylakoid lumen of chloroplasts. Isolation and characterization

The chloroplast compartment enclosed by the thylakoid membrane, the "lumen," is poorly characterized. The major aims of this work were to design a procedure for the isolation of the thylakoid lumen which could be generally used to characterize lumenal proteins. The preparation was a stepwise procedure in which thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, and following Yeda press fragmentation the lumenal content was recovered in the supernatant following centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins specific for the different chloroplast compartments. Quantitative immunoblot analyses showed that the recovery of soluble lumenal proteins was 60-65% (as judged by the presence of plastocyanin), whereas contamination with stromal enzymes was less than 1% (ribulose-bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were recovered in the lumenal fraction, of which several were identified for the first time. Enzymatic measurements and/or amino-terminal sequencing revealed the presence of proteolytic activities, violaxanthin de-epoxidase, polyphenol oxidase, peroxidase, as well as a novel prolyl cis/trans-isomerase.     

A mechanism for the formation of inside-out membrane vesicles. Preparation of inside-out vesicles from membrane-paired randomized chloroplast lamellae

Inside-out thylakoid membrane vesicles can be isolated by aqueous polymer two-phase partition of Yeda press-fragmented spinach chloroplasts (Andersson, B. and Akerlund, H.-E. (1978) Biochim. Biophys. Acta 503, 462-472). The mechanism for their formation has been investigated by studying the yield of inside-out vesicles after various treatments of the chloroplasts prior to fragmentation. No inside-out vesicles were isolated during phase partitioning if the chloroplasts had been destacked in a low-salt medium prior to the fragmentation. Only in those cases where the chloroplast lamellae had been stacked by cations of membrane-paired by acidic treatment did we get any yield of inside-out vesicles. Thus, the intrinsic properties of chloroplast thylakoids seem to be such that they seal into right-side out vesicles after disruption unless they are in an appressed state. This favours the following mechanism for the formation of inside-out thylakoids. After press treatment, a ruptured membrane still remains appressed with an adjacent membrane. Resealing of such an appressed membrane pair would result in an inside-out vesicle. If the compartmentation of chloroplast lamellae into appressed grana and unappressed stroma lamellae is preserved by cations before fragmentation, the inside-out vesicles are highly enriched in photosystem II. This indicates a granal origin which is consistent with the proposed model outlined. Inside-out vesicles possessing photosystem I and II properties in approximately equal proportions could be obtained by acid-induced membrane-pairing of chloroplasts which had been destacked and randomized prior to fragmentation. Since this new preparation of inside-out thylakoid vesicles also exposes components derived from the stroma lamellae it complements the previous preparation. It is suggested that fragmentation of paired membrane followed by phase partitioning should be a general method of obtaining inside-out vesicles from membranes of various biological sources.     

Role of signal peptides in targeting of proteins in cyanobacteria

Proteins of cyanobacteria may be transported across one of two membrane systems: the typical eubacterial cell envelope (consisting of an inner membrane, periplasmic space, and an outer membrane) and the photosynthetic thylakoids. To investigate the role of signal peptides in targeting in cyanobacteria, Synechococcus sp. strain PCC 7942 was transformed with vectors carrying the chloramphenicol acetyltransferase reporter gene fused to coding sequences for one of four different signal peptides. These included signal peptides of two proteins of periplasmic space origin (one from Escherichia coli and the other from Synechococcus sp. strain PCC 7942) and two other signal peptides of proteins located in the thylakoid lumen (one from a cyanobacterium and the other from a higher plant). The location of the gene fusion products expressed in Synechococcus sp. strain PCC 7942 was determined by a chloramphenicol acetyltransferase enzyme-linked immunosorbent assay of subcellular fractions. The distribution pattern for gene fusions with periplasmic signal peptides was different from that of gene fusions with thylakoid lumen signal peptides. Primary sequence analysis revealed conserved features in the thylakoid lumen signal peptides that were absent from the periplasmic signal peptides. These results suggest the importance of the signal peptide in protein targeting in cyanobacteria and point to the presence of signal peptide features conserved between chloroplasts and cyanobacteria for targeting of proteins to the thylakoid lumen.     

Photoreceptor membrane assymetric structure in separated phase state

The structure of unbleached bovine retinal rod photoreceptor membranes isolated in ficoli density gradient has been studied by means of small-angle X-ray diffraction methods. Samples were prepared in the form of thin multilamellar films of photoreceptor membranes in phase-separated state induced by partial dehydration. Diffraction data were collected using diffractometer with linear position-sensitive detector. Phase signs of structure amplitude were determined by method [7] and membrane lamellar electron density distribution was calculated at 1, 7 nm resolution. The results obtained showed photoreceptor membranes isolated in ficoli density gradient to have asymmetric structure which differed from that of photoreceptor membranes isolated in sucrose density gradient [1]. The asymmetry observed may be accounted for different content of lipid L alpha and L beta phases in cytoplasmic and intradisk membrane layers. It may be assumed that ficoli helps to support membrane native structure.     

Small-angle X-ray scattering from mitochondria

X-ray (CuKalpha) scattering curves of rat liver mitochondria are characterized by continuously decreasing intensity from 0.5 to 5 mrad and a broad maximum centered near 20 mrad. The condensed-to-orthodox morphological transition of the inner membranes of intact mitochondria causes a dramatic decrease in scattering at very small angle and a marked shift of the 20 mrad maximum to smaller angle. A similar small-angle scattering maximum is observed with inner mitochondrial membrane fractions prepared by digitonin treatment and osmotic shock/step gradient centrifugation procedures. However, the small-angle X-ray scattering curves of mitochondria after acetone treatment and osmoticlysis/sonication are essentially continuous. These characteristics of mitochondrial X-ray scattering are discussed in terms of known structural features of the organelle.     

Occupational allergic contact dermatitis caused by epoxy diacrylate in ultraviolet-light-cured paint, and bisphenol A in dental composite resin

Calcium ion-dependent reactivation of O2 evolution activity has been investigated in Hydrilla verticillata thylakoid preparations. Washing the thylakoids in calcium-free buffer or calcium-free buffer containing 1.5 M NaCl or 1.5 M NaCl plus 20% methanol, reversibly inhibited O2 evolution activity. The activity was restored on addition of calcium as calcium chloride and partially by strontium chloride. Immobilization of thylakoids with glutaraldehyde (GA) arrested the loss in O2 evolution activity caused by calcium-free high salt washing. However, calcium sensitivity was discernible in GA immobilized thylakoids subjected to calcium-free high salt washing. Since glutaraldehyde checks the loss of extrinsic thylakoid polypeptides due to washing, it is assumed that the calcium ion has regulatory functions in the photosynthetic electron transport, besides its interaction with thylakoid proteins.     

Effect of modification of the smoking habit on lung function

Envelope- and stroma-free thylakoid membranes of Vicia faba chloroplasts were incubated with trypsin or pronase for several hours. The indigestible residue was analysed by polyacrylamide gel electrophoresis. Trypsinization resulted in a complete digestion of all proteins with the exception of the pigment-protein complexes as well as a polypeptide not yet characterized. Yet, as compared with untreated material, Complex II was found to have higher electrophoretic mobility. Electron-microscopic studies illustrate that the indigestible residue still has a preserved membrane structure. Disintegration of the thylakoid membranes by sodium dodecyl sulfate followed by trypsinization also resulted in the two complexes while all the other proteins were found to be digested. However, after removal of the lipids the protein moieties of the complexes proved to be easily digestible. From these results it is concluded that pigment-protein interaction may be an important factor in maintaining a conformation rather resistant to perturbants and proteases. In contrast to trypsin, pronase completely digested the polypeptides of the thylakoid membranes including the protein moieties of the pigment-protein complexes leaving an amorphous lipid mass. The results support the assumption that the complexes are necessary to maintain the membrane structure.     

X-ray and neutron scattering density profiles of the intact human red blood cell membrane

Hemoglobin-free human red blood cell membranes have been prepared with glutaraldehyde to maintain an intact structure on partial dehydration. Treatment of resealed ghosts with poly(L-lysine) produced an essentially constant structural unit and permitted correlation of electron microscopy results with x-ray and neutron diffraction profiles. These profiles provide detailed information, for the intact membrane, on the location and relative distribution of lipids and proteins.     

Comparative study of membranes of Streptococcus faecalis and Micrococcus lysodeikticus

A comparative study of ultrastructure and IR-spectroscopy of osmotic shock membranes from cells of glycolyzing (Streptococcus faecalis) and respiring (Micrococcus lysodeikticus) bacteria, was made. The S. faecalis and M. lysodeikticus membranes differ in their cross-section. Treatment of the preliminary washed membranes of S. faecalis and M. lysodeikticus with a low ionic strength solution removes 40% and 70% of their proteins respectively, decreases the membrane cross-section but does not change their fracture faces. Pre-cooling of the membrane suspensions within the temperature range of +5 degrees-10 degrees results in the appearance of large smooth areas on S. faecalis membrane fracture faces, but does not affect the ones of M. lysodeikticus membrane. Treatment of the washed suspensions with Triton X-100 results in the appearance of drastic changes of S. faecalis membrane fracture faces and does not change the fracture faces of M. lysodeikticus membranes; treatment by the detergent does not alter the IR-spectroscopy of membranes of both bacteria. Treatment of S. faecalis and M. lysodeikticus membranes with high temperature irreversibly changes the structure of 20% and 40% of protein components respectively,, but does not affect the distribution of the subparticles on their fracture faces. It is assumed that the differences found are determined by the composition of lipid components of the membranes studied and that the amount of proteins closely bound with lipids in the membranes of S. faecalis is likely to be greater than that of M. lysodeikticus membranes.     

Interaction of gangliosides with proteins depending on oligosaccharide chain and protein surface modification

By using neutron and synchrotron x-ray small-angle scattering techniques, we investigated the process of the complexation of gangliosides with proteins. We treated monosialoganglioside (G(M1)), disialoganglioside (G(D1a)), and a mixture of G(M1)/G(D1a). Proteins used were bovine serum albumins whose surfaces were modified with different sugars (deoxy-D-galactose, deoxy-L-fucose, deoxymaltitol, and deoxycellobiitol), which were used as model glycoproteins in a membrane. We found that the complexation of gangliosides with albumins greatly depends on the combination of ganglioside species and protein surface modification. With a varying protein/ganglioside ratio in a buffer solution at pH 7, the complexation of G(M1) or G(D1a) with albumins modified by monosaccharides appears to be less destructive for ganglioside aggregate structures in forming large complexes; the complexation of G(D1a) with the albumins modified by disaccharides induces the formation of complexes with a dimeric structure; and the complexation of G(M1) with albumins modified by disaccharides, to form small complexes, is very destructive. The present results show a strong dependence of the interaction between ganglioside and protein on the characteristics of the ganglioside and protein surface, which would relate to a physiological function of gangliosides, such as a function regulating the receptor activity of glycoproteins in a cell membrane.     

Existence of a flat phase in red cell membrane skeletons

Biomolecular membranes display rich statistical mechanical behavior. They are classified as liquid in the absence of shear elasticity in the plane of the membrane and tethered (solid) when the neighboring molecules or subunits are connected and the membranes exhibit solid-like elastic behavior in the plane of the membrane. The spectrin skeleton of red blood cells was studied as a model tethered membrane. The static structure factor of the skeletons, measured by small-angle x-ray and light scattering, was fitted with a structure factor predicted with a model calculation. The model describes tethered membrane sheets with free edges in a flat phase, which is a locally rough but globally flat membrane configuration. The fit was good for large scattering vectors. The membrane roughness exponent, zeta, defined through h alpha L zeta, where h is the average amplitude of out-of-plane fluctuations and L is the linear membrane dimension, was determined to be 0.65 +/- 0.10. Computer simulations of model red blood cell skeletons also showed this flat phase. The value for the roughness exponent, which was determined from the scaling properties of membranes of different sizes, was consistent with that from the experiments.     

Small-angle neutron scattering investigation of creep damage in type 304 stainless steel and alloy 800

Creep damage accumulation in fully precipitated type 304 stainless steel and alloy 800 was investigated using small-angle neutron scattering. The small-angle scattering decreased initially and then slowly increased during the intermediate stages of creep. The scattering is thought to be dominated by stress assisted coarsening of carbide precipitates during the early stages of creep. This result and the direct observation of cavities by transmission electron microscopy indicate that at least two damage mechanisms, precipitate coarsening and cavitation, were occurring simultaneously. Coarsening dominates the small-angle scattering measurements during the early stages of creep, but cavitation appears to be detectable during the intermediate stages.     

Purification and properties of a novel chloroplast stromal peptidase. Processing of polyphenol oxidase and other imported precursors

Polyphenol oxidases (PPOs) are nuclear-encoded chloroplast proteins that are targeted to the thylakoid lumen by a bipartite presequence. The N-terminal part of this sequence is removed by a stromal processing peptidase (SPP), and the resulting intermediate is translocated across the thylakoid and processed to the mature protein. A 4800-fold-purified SPP processed a PPO precursor (pPPO) at a site identical to that occurring in organelle. The in vitro product of SPP action on pPPO was further processed and translocated by thylakoids. This SPP processed other precursors but was inactive toward those of light-harvesting chlorophyll binding proteins. The enzyme appeared to be a metalloendopeptidase, like previously reported SPPs. However, it differed in substrate specificity, apparent size, and, most significantly, cleavage site of pPPO. Whereas the processing sites of lumen proteins determined so far were relatively distant from the hydrophobic core of the thylakoid targeting domain, pPPO was cleaved immediately before this domain. Cleavage removed the twin arginine motif characteristic of thylakoid targeting domains of lumen proteins, which are translocated by the DeltapH-dependent pathway. The possible significance of these observations to PPO translocation mechanism is discussed. It is suggested that several SPPs may exist in chloroplasts with preferences for different subsets of precursors.     

Anxiety and alcohol abuse in patients in treatment for depression

Oxygen transport in thylakoid membranes of spinach chloroplasts (Spinacia oleracea) has been studied by observing the collisions of molecular oxygen with spin labels, using line broadening electron paramagnetic resonance (EPR) spectroscopy. Stearic acid spin labels were used to probe the local oxygen diffusion-concentration product. The free radical moiety was located at various distances from the membrane surface, and collision rates were estimated from linewidths of the EPR spectra measured in the presence and absence of molecular oxygen. The profile of the local oxygen diffusion-concentration product across the membrane determined at 20 degrees C demonstrates that this product, at all membrane locations, is higher than the value measured in water. From the profile of the oxygen diffusion-concentration product, the membrane oxygen permeability coefficient has been estimated using the procedure developed earlier (W.K. Subczynski, J.S. Hyde, A. Kusumi, Proc. Natl. Acad. Sci. USA 86 (1989) 4474-4478). At 20 degrees C, the oxygen permeability coefficient for the lipid portion of the thylakoid membrane was found to be 39.5 cm s-1. This value is 20% higher than the oxygen permeability coefficient of a water layer of the same thickness as the thylakoid membrane. The high permeability coefficient implies that the oxygen concentration difference across the thylakoid membrane generated under the illumination of the leaf by saturating actinic light is negligible, smaller than 1 microM.     

Cavity nucleation at grain boundary ledges

The kinetics of cavity nucleation at grain-boundary ledges is examined for ceramics subjected to compressive loads. By considering grain boundary sliding and diffusion as concurrent processes, the analysis shows that sliding of faceted grain boundaries can induce time-dependent stress concentrations of sufficient magnitude to cause cavity nucleation at the ledges. The transient stress concentration has been found to depend on the ledge height and spacing, and to decrease rapidly with time and with distance from the ledge. Whether or not cavity nucleation occurs at the ledge depends on the local stress, the shape of the critical nucleus, and the characteristic time for cavity formation. The theoretical results are discussed in conjunction with nucleation rate measurements obtained for a sintered alumina using small-angle neutron scattering.     

Glassy Carbon as an Absolute Intensity Calibration Standard for Small-Angle Scattering

Absolute calibration of small-angle scattering (SAS) intensity data (measured in terms of the differential scattering cross section per unit sample volume per unit solid angle) is essential for many important aspects of quantitative SAS analysis, such as obtaining the number density, volume fraction, and specific surface area of the scatterers. It also enables scattering data from different instruments (light, X-ray, or neutron scattering) to be combined, and it can even be useful to detect the existence of artifacts in the experimental data. Different primary or secondary calibration methods are available. In the latter case, absolute intensity calibration requires a stable artifact with the necessary scattering profile. Glassy carbon has sometimes been selected as this intensity calibration standard. Here we review the spatial homogeneity and temporal stability of one type of commercially available glassy carbon that is being used as an intensity calibration standard at a number of SAS facilities. We demonstrate that glassy carbon is sufficiently homogeneous and stable during routine use to be relied upon as a suitable standard for absolute intensity calibration of SAS data.