Kinetics of the appearance of mRNA for light-harvesting chlorophyll a/b protein in polysomes of barley

Polysomes from dark-grown and illuminated barley seedlings were translated in cell-free systems. The translation products reacting with the antibody against the light-harvesting chlorophyll a/b protein (LHCP) were analyzed by polyacrylamide gel electrophoresis. It was found that, in addition to the precursor protein of LHCP, a product was obtained that co-migrated with the mature protein. Furthermore, the results show that the light-induced proly(A)RNA for LHCP is integrated into the polysomal complex without delay, indicating that the integration of LHCP into the membrane is controlled at a higher level of gene expression.     

Protein interactions with lipid bilayers: The channels of kidney plasma membrane proteolipids

Summary Proteolipids extracted from bovine kidney plasma membrane induce irreversible changes in the electrical properties of lipid bilayers formed from diphytanoyl phosphatidylcholine. The interaction with the proteolipid produces channels which are cation selective. At low protein concentrations (i.e., <0.6 g/ml), the single-channel conductance is approximately 10 pS in 100mm KCl and 3 pS in 100mm NaCl. In the presence of protein concentrations above 1 g/ml, another population of channels appears. These channels have a conductance of about 100 pS in 100mm KCl and 30 pS in 100mm NaCl. Further, these channels are voltage dependent in KCl, closing when the voltage is clamped at values 30 mV. The steady-state membrane conductance, measured at low voltages, was found to increase proportional to a high power (2–3) of the proteolipid concentration present in one of the aqueous phases. In 100mm NaCl, the conductance increases at protein concentrations above 5 g/ml, whereas in 100mm KCl in increases at protein concentrations above 0.6 g/ml. These measurements indicate that the higher steady-state conductance observed in KCl at a given proteolipid concentration in a multi-channel membrane presumably results because more channels incorporate in the presence of KCl than in the presence of NaCl.The two major fractions which comprise the proteolipid complex were also tested on bilayers. It was found that both fractions are required to produce the effects described.     

Benzyladenine induces the appearance of LHCP-mRNA and of the relevant protein in dark-grown excised watermelon cotyledons

Cotyledons were excised from imbibed watermelon seeds, grown for 4 days in darkness on water or 10 M benzyladenine (BA) and then tested for the presence of the light-harvesting chlorophyll a/b protein (LHCP) and its mRNA. LHCP was assayed immunologically by western blotting of SDS gels: the protein was present in plastids, but it was not recovered with the thylakoid fraction. Antibodies directed against LHCP precipitated a 32 kDa polypeptide from translation products of poly(A) RNA of cotyledons only if these had been grown on BA. Taken together the data suggest that in absence of light cytokinins are necessary for the maintenance of a detectable level of LHCP-mRNA as well as for synthesis of the protein.     

Submicroscopic structure of the membranous labyrinth

Summary Investigations were performed by light and electron microscope on the basilar membrane, limbus spiralis and spiral ligament.These different parts continue one into the other and make up a single morphological and functional structure which may be called the supporting structure of Corti's organ (s.s.C.o.).It is formed by a tissue the components of which are the cells and an intercellular substance in which are arranged the capillary vessels.The cells can be classed in two groups, the first consisting of the cells proper (basilar membrane, limbus spiralis and spiral ligament cells) which present structural changes parallel with the growth mechanism of the intercellular substance; the second of the cochlear duct covering cells (Corti's organ cells, inner and outer spiral sulcus cells, interdental cells, stria vascularis cells).The intercellular substance is organised in laminae, fibrolaminae, bundles and microscopic fibers composed of filaments with an intervening ground substance.The filaments have a diameter ranging from 85 to 105 Å. Topochemical tests with polarised light microscope, enzymatic tests, diffractographic and chemical analyses suggest that these filaments unquestionably consist of protein material which have nothing to do with collagen or elastic fibers. Perhaps it may be classed in the K.E.M.F. group.The ground substance generally appear anhistous and transparent but in some parts of the basilar membrane it presents a cottony appearance.The possible different hypotheses about the classification of the s.s.C.o. tissue are discussed.The quantity and architecture of the cells and the intercellular substance vary appreciably in the basilar membrane, limbus spiralis and spiral ligament, which are examined in detail one by one.The demonstration that the s.s.C.o. is formed of a tissue possessing an intercellular substance containing filamentous scleroproteins clearly corroborates the theory that is performs supporting activity in respect of Corti's organ. The term supporting structure of Corti's organ is based on this interpretation.Research financed by C.N.R. grant.     

Relationship of nuclear membranes with filaments and microtubules

Summary In certain HeLa cells characteristic aggregates of cytoplasmic filaments, microfilaments and microtubules were found which preferentially encompass the nucleus. These structures are intimately associated with the membranes of the nuclear envelope and the endoplasmic reticulum as well as with ribosomes. Starting from this observation, a review is presented which demonstrates the nuclear membranes generally as being a type of membrane with a close relationship to proteinaceous structures of the actin-like type such as microtubules and (micro)filaments. As an explanation for the phenomena observed, it is hypothesized that the formation of such tubules and filaments is nucleated at cytomembraneous surfaces and/or that membrane (and possibly also ribosomal) material can be transformed to such tubular or filamentous structures. The concept of cocrystallization between membranes and the diverse tubular and filamentous categories and of a transformation of membrane protein moieties into such structures is thought to provide a basis for explaining many structural phenomena involving membranes and structures of actin-like proteins.The author gratefully acknowledges the careful technical assistance of MissSigrid Krien and MissMarianne Winter and valuable discussions with his colleagues, Drs. H.Falk, H.Kleinig, U.Scheer, as well as with Drs. M.Moses (Duke University), D. J.Morré (Purdue University), and K.Wolff (University of Vienna). The work was supported by the Deutsche Forschungsgesellschaft.     

Hexagonally ordered “rosettes” of particles in the plasma membrane ofMicrasterias denticulata Bréb. and their significance for microfibril formation and orientation

Summary Cells ofMicrasterias denticulata Bréb. at the stage of secondary wall formation have been studied by freeze-etching. It was found that the plasma membrane exhibits oval areas in which arrays of membrane particles occur. These particles form rosettes which are arranged in a hexagonally ordered lattice with a center to center spacing of 25 nm. Nearly the same periodicities can be found between microfibrils. It is concluded that the rosettes probably together with the thickened area of the plasma membrane below them represent the apparatus for the production and orientation of microfibrils. The hypothesis suggesting the incorporation of membrane templates functional in microfibril formation, originally advanced byKiermayer andDobberstein (1973) has received further support.     

Thylakoid-protein phosphorylation during the life cycle of Scenedesmus obliquus in synchronous culture

The phosphorylation of thylakoid proteins, which comprise apoproteins of the light-harvesting chlorophyll a/b-protein complex (LHCP), was investigated in vivo and in vitro during the development of Scenedesmus obliquus in synchronous cultures. The in-vitro and in-vivo protein phosphorylation exhibited a maximum activity in cells with maximum photosynthetic capacity (8th hour) and miximum activity in cells with minimum photosynthetic capacity (16th hour). The major phosphorylated polypeptides in vivo were the 24/25-kDa and 28–30-kDa apoprotein of the LHCP, a protein of about 32 kDa, and some smaller polypeptides within the range 10 to 20 kDa. In vitro, the main phosphoproteins were the 28–30-kDa apoprotein and the protein characterized by an apparent molecular weight of 32 kDa. Pulse-chase experiments in vivo established that the latter had the fastest radioactivity turnover of the thylakoidal phosphoproteins.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP light-harvesting chlorophyll a/b-protein complex - PSII photosystem II Dedicated to Prof. Erwin Bünning on the occasion of his 80^th birthday     

Expression of nuclear genes as affected by treatments acting on the plastids

In a preceding paper (Oelmüller and Mohr 1986, Planta 167, 106–113) it was shown that in the cotyledons of the mustard (Sinapis alba L.) seedling the integrity of the plastid is a necessary prerequisite for phytochrome-controlled appearance of translatable mRNA for the nuclear-encoded small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase and the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCP). It was concluded that a signal from the plastid is essential for the expression of nuclear genes involved in plastidogenesis. The present study was undertaken to characterize this postulated signal. Chloramphenicol, an inhibitor of intraplastidic protein synthesis and Norflurazon, an inhibitor of carotenoid synthesis (to bring about photooxidative sensitivity of the plastids) were applied. We obtained the following major results. (i) After a brief period of photooxidative damage a rapid decrease of the above translatable mRNAs was observed. Conclusion: the signal is short-lived and thus required continually. (ii) Once the plastids became damaged by photooxidation, no recovery with regard to nuclear gene expression was observed after a transfer to non-damaging light conditions. Conclusion: even a brief period of damage suffices to prevent production of the signal. (iii) Chloramphenicol inhibited nuclear gene expression (SSU, LHCP) and plastidic development when applied during the early stages of plastidogenesis. Once a certain stage had been reached (between 36–48 h after sowing at 25° C) nuclear gene expression became remarkably insensitive toward inhibition of intraplastidic translation. Conclusion: a certain developmental stage of the plastid must be reached before the signal is released by the plastid. (iv) Under the growth conditions we adopted in our experiments the plastids in the mesophyll cells of mustard cotyledons developed essentially between 36 and 120 (-144) h after sowing. Only during this period could translatable mRNAs for SSU and LHCP be detected. Conclusion: the signal is released by the plastids only during this time span.Abbreviations CAP Chloramphenicol (D-threo) - cFR continuous far-red light - FR far-red light (3.5 W·m^-2) - GPD glyceraldehyde-3-phosphate dehydrogenase - LHCP light-harvesting chlorophyll a/b-binding protein of photosystem II - LSU large subunit of RuBPCase - MDH malate dehydrogenase - NF Norflurazon - NIR nitrite reductase - Pfr physiologically active form of phytochrome - R red light (6.8 W·m^-2) - RG9-light long-wavelength far-red light (10 W·m^-2) - RuBPCase ribulose-1,5-bisphosphate carboxylase - SSU small subunit of RuBPCase - WL_s strong white light (28 W·m^-2) - photoequilibrium of phytochrome at wavelength      

Pullulanase,an enzyme of starch catabolism,is associated with the outer membrane of Klebsiella

Late-log phase cells of Klebsiella sp. 5246 could be converted into spheroplasts with a yield of better than 90% by ethylenediamine tetraacetate/lysozyme treatment in osmotically stabilizing media. Membrane fragments obtained after ultrasonication of spheroplasts were separated by centrifugation to sedimentation equilibrium on a sucrose density gradient. A light membrane fraction with a buoyant density of 1.17±0.02g/cm³ was sought and found to contain the enzymes NADH oxidase, succinate dehydrogenase and D-lactate dehydrogenase. A heavy membrane fraction having a buoyant density of 1.23 ±0.01g/cm³ was characterized by phospholipase A₁ activity and lipopolysaccharide content. By analogy to other gram-negative bacteria, the light and the heavy fraction were assigned, respectively, to the cytoplasmic and the outer membrane of Klebsiella sp. 5246.The organism produced pullulanase in a cellbound form during the exponential phase of growth on soluble starch. Pullulanase was localized exclusively on the outer membrane. Pullulanase is the second protein of the outer membrane with defined enzyme function to become known among gram-negative bacteria, the other one being phospholipase A₁.What had been inferred from physiological studies of growth characteristics on various carbon sources can now be proven directly: Pullulanase implicated in the utilization of branched -glucans in Klebsiella is capable of acting on macromolecular substrates in the environment of the cell by virtue of its association with the outer membrane.Non-Standard Abbreviations EDTA ethylenediamine tetraacetate - SDS sodium dodecyl sulphate - OD optical density List of Enzymes EC 3.2.1. 23 -galactosidase or -D-galactoside galactohydrolase - EC 1.1.1.28 D-lactate dehydrogenase or D-lactate: NAD⁺ oxidoreductase - EC 3.2.1.17 lysozyme or mucopeptide N-acetylmuramoylhydrolase - EC 2.4.1.1 maltodextrin phosphorylase or 1,4--D-glucan: orthophosphate -glucosyltransferase - EC 1.6.99.3 NADH oxidase or NADH: (acceptor) oxidoreductase - EC 3.1.1.32 phospholipase A₁ or phosphatide 1-acylhydrolase - EC 3.2.1.41 pullulanase or pullulan 6-glucanohydrolase - EC 1.3.99.1 succinate dehydrogenase or succinate: (acceptor) oxidoreductase     

Effects of membrane potential on Na cotransports in eel intestinal brush-border membrane vesicles: Studies with a fluorescent dye

Summary The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3-diethylthiacarbocyanine iodide (DiS-C₂(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking in vivo conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andK _app values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.     

Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris

Summary A vanadate-sensitive H⁺-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H⁺-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H⁺ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H⁺ transport activity was inhibited half-maximally between 1 and 5 M vanadate (Na₃VO₄) and nearly abolished by 100 M vanadate. ATPase activity of native plasma membrane showed aK _i for vanadate inhibition of 9.5 M, and was inhibited up to 80% by 15 to 20 M vanadate (Na₃VO₄). ATPase activity of the reconstituted vesicles showed aK _i of 2.6 M for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.     

Inhibition of anion permeability by amphiphilic compounds in human red cell: Evidence for an interaction of niflumic acid with the band 3 protein

Summary In human erythrocyte, permeability to the anion is instantaneously, reversibly, and noncompetitively inhibited by the nonsteroidal anti-inflammatory drug, niflumic acid. The active form of this powerful inhibitor (I ₅₀=6×10^–7 m) is the ionic form. We demonstrated that: (i) The binding of niflumic acid to the membrane of unsealed ghosts shows one saturable and one linear component over the concentration range studied. The saturable component vanishes when chloride transport is fully inhibited by covalently bound 4-acetamido-4-isothiocyano stilbene-2,2-disulfonic acid (SITS). Our estimate of these SITS protectable niflumate binding sites (about 9×10⁵ per cell) agrees with the number of protein molecules per cell in band 3. These sites are halfsaturated with 10^–6 m niflumic acid, a concentration very close toI ₅₀. (ii) Niflumic acid inhibits the binding reaction of SITS with anion controlling transport sites. These results indicate that niflumic acid and SITS are mutually exclusive inhibitors, suggesting that niflumic acid interacts with the protein in band 3.Niflumic acid also decreases glucose and ouabain-insensitive sodium permeabilities. However, these effects are produced at a very high concentration of niflumic acid (in millimolar range), suggesting unspecific action, possibly through lipid phase.     

Phosphorylation of heavy sarcoplasmic reticulum vesicles: Identification and characterization of three phosphorylated proteins

Summary Heavy sarcoplasmic reticulum vesicles derived from the terminal cisternae of the sarcoplasmic reticulum have been shown to contain endogenous protein kinase activity and associated substrate proteins. Heavy vesicles were phosphorylated at room temperature in 5mm MgCl₂, 1mm EGTA, 10mm HEPES (pH 7.4) and 10 m -³²P-ATP.³²P-phosphoproteins were determined by sodium dodecyl sulphate gel electrophoresis and autoradiography. In the absence of ethylene glycol bis (-aminoethyl ether) N,N,N,N-tetraacetic acid (EGTA), there was little phosphorylation due to the high level of ATPase activity. Phosphorylation of three proteins of 64,000 daltons (E1), 42,000 daltons (E2), and 20,000 daltons (E3) was observed in the presence of 1mm EGTA. Phosphorylation of these proteins wascAMP-independent, hydroxylamine-resistant, and was seen without the addition of protein kinase. In the presence of HgCl₂ (2.5mm) or sodium deoxycholate (1%) no protein phosphorylation was observed. ProteinE1 was heavily phosphorylated in the presence of 200mm KCl, while its phosphorylation was inhibited by 20 m sodium dantrolene, an inhibitor of Ca²⁺ release. PhosphoproteinE3 was found in light and heavy sarcoplasmic reticulum vesicles whileE1 andE2 were found only in heavy vesicles. The phosphoproteinE2 had the properties of an intrinsic membrane protein while the proteinE1 bejaved as an extrinsic membrane protein. ProteinsE2 andE3 corresponded in mobility to minor sarcoplasmic reticulum proteins whileE1 had the same mobility as calsequestrin. The presence of high calcium (5mm) during electrophoresis caused calsequestrin to run at a lower molecular weight (56,000 instead of 64,000 daltons), and correspondingly the phosphoproteinE1 ran at a lower molecular weight. Finally, calsequestrin purified by a double gel electrophoresis method has been shown to be phosphorylated.     

Isolation and characterisation of the Photosystem two reaction centre complex from a double mutant of Chlamydomonas reinhardtii

A rapid procedure has been developed for the isolation of the photosystem two reaction centre complex (PS II RC) from a double mutant of Chlamydomonas reinhardtii, F54-14, which lacks the Photosystem one complex and the chloroplast ATPase. Thylakoid membranes are solubilised with 1.5% (w/v) Triton X-100 and the PS II RC purified by anion-exchange chromatography using TSK DEAE-650(S) (Merck). The complex has a pigment stoichiometry of approximately six chlorophyll a: two pheophytin a: one cytochrome b-559: one to two -carotene. It photoaccumulates reduced pheophytin and oxidised P680 in the presence of sodium dithionite and silicomolybdate, respectively. Immunoblotting experiments have confirmed the presence of the D1 and D2 polypeptides in this complex. The -subunit of cytochrome b-559 was identified by N-terminal sequencing. Comparison of the complex with the PS II RC from pea using SDS-polyacrylamide gel electrophoresis showed that their polypeptide compositions were similar. However, the -subunit of cytochrome b-559 from C. reinhardtii has a lower apparent molecular weight than the pea counterpart whereas the -subunit is larger.Abbreviations DM n-dodecyl -d-maltoside - RC reaction centre - SiMo silicomolybdate, SiMo₁₂O₄₀ ^4– - TAP Tris-acetate-phosphate     

Restoration of membrane excitability of squid giant axons by reagents activating tyrosine-tubulin ligase

Summary Using squid giant axon, an experimental survey was performed on restoration of the membrane excitability which had been partially suppressed. Among reagents examined, a combination of 400mm KF, 50 m tyrosine, 1mm ATP, 1mm Mg ions and 5 m cAMP was found to induce the restoration of the excitability to a large extent. Further addition of a small amount of either porcine brain microtubule proteins or the squid axoplasm was found to support complete restoration. The experiments suggest that tubulin-tyrosine ligase contained in the porcine brain microtubule protein fraction or the squid axoplasm maintains the coupling between cytoskeletal structures and the plasma membrane.     

The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

Modulation of water and urea transport in human red cells: Effects of pH and phloretin

Summary It has previously been shown by Macey and Farmer (Biochim. Biophys. Acta 211:104–106, 1970) that phloretin inhibits urea transport across the human red cell membrane yet has no effect on water transport. Jennings and Solomon (J. Gen. Physiol. 67:381–397, 1976) have shown that there are separate lipid and protein binding sites for phloretin on the red cell membrane. We have now found that urea transport is inhibited by phloretin binding to the lipids with aK ₁ of 25±8 m in reason-able agreement with theK _D of 54±5 m for lipid binding. These experiments show that lipid/protein interactions can alter the conformational state of the urea transport protein. Phloretin binding to the protein site also modulates red cell urea transport, but the modulation is opposed by the specific stilbene anion transport inhibitor, DIDS (4,4-diisothiocyano-2,2-stilbene disulfonate), suggesting a linkage between the urea transport protein and band 3. Neither the lipid nor the protein phloretin binding site has any significant effect on water transport. Water transport is, however, inhibited by up to 30% in a pH-dependent manner by DIDS binding, which suggests that the DIDS/band 3 complex can modulate water transport.     

Chloride permeability in human red cells: Influence of membrane protein rearrangement resulting from ATP depletion and calcium accumulation

Summary As 15% of band 3 protein, the assumed chloride channel, is associated with spectrin, the major peripheral protein of a lattice located at the red cell membrane-cytosol interface, the present study was undertaken to evaluate whether a rearrangement of the lattice modifies the functional property of band 3 protein. Such a rearrangement was modulated by depletion of cell ATP and/or by accumulation of Ca²⁺ ions within the cell.ATP depletion induces an inhibition of the electroneutral one-for-one chloride exchanges. Neither the modification of red cell morphology due to ATP depletion (discocyte-echinocyte transformation) nor a direct effect of the decrease in internal ATP level can account for this inhibition. On the other hand, it seems reasonable to consider that inhibition is related to the changes in membrane protein organization (formation of heteropolymers) induced by the decrease in ATP level. But it does not appear that the degree of inhibition is modified when this altered assembly of membrane protein is stabilized by disulfide linkages.Accumulation of Ca²⁺ ions in the cell at a relatively low concentration (10m range) inhibits chloride exchange without apparent modification of the assembly of membrane proteins. This effect of calcium on chloride exchanges is speculatively denoted as a direct effect of calcium.Calcium loading of fresh red cells at higher concentrations (500 to 1000 m) obtained by use of the ionophore A23187 induces a very strong inhibition of chloride exchanges. In this case, inhibition can be reasonably accounted for by two simultaneous effects of calcium: a direct effect which explains half of the inhibition and an indirect effect due to the formation of membrane protein complexes stabilized by covalent crosslinkages (activation by Ca²⁺ ions of a transglutaminase).It is interesting to note that intracellular calcium, whatever the level, inhibits electroneutral exchanges of chloride but increases net chloride movements.     

A stromal protein factor maintains the solubility and insertion competence of an imported thylakoid membrane protein

The light-harvesting chlorophyll a/b protein (LHCP) is an approximately 25,000-D thylakoid membrane protein. LHCP is synthesized in the cytosol as a precursor and must translocate across the chloroplast envelope before becoming integrally associated with the thylakoid bilayer. Previous studies demonstrated that imported LHCP traverses the chloroplast stroma as a soluble intermediate before thylakoid insertion. Here, examination of this intermediate revealed that it is a stable, discrete approximately 120,000-D species and thus either an LHCP oligomer or a complex with another component. In vitro-synthesized LHCP can be converted to a similar form by incubation with a stromal extract. The stromal component responsible for this conversion is proteinaceous as evidenced by its inactivation by heat, protease, and NEM. Furthermore, the conversion activity coelutes from a gel filtration column with a stromal protein factor(s) previously shown to be necessary for LHCP integration into isolated thylakoids. Conversion of LHCP to the 120-kD form prevents aggregation and maintains its competence for thylakoid insertion. However, conversion to this form is apparently not sufficient for membrane insertion because the isolated 120-kD LHCP still requires stroma to complete the integration process. This suggests a need for at least one more stroma-mediated reaction. Our results explain how a hydrophobic thylakoid protein remains soluble as it traverses the aqueous stroma. Moreover, they describe in part the function of the stromal requirement for insertion into the thylakoid membrane.     

Membrane stretch augments the cardiac muscarinic K+ channel activity

Arachidonic acid has been shown to activate K⁺-selective, mechanosensitive ion channels in cardiac, neuronal and smooth muscle cells. Since the cardiac G protein (G _K )-gated, muscarinic K⁺ (K_ACh) channel can also be activated by arachidonic acid, we investigated whether the K_ACh channel was also sensitive to membrane stretch. In the absence of acetylcholine (ACh), K_ACh channels were not active, and negative pressure failed to activate these channels. With ACh (10 m) in the pipette, applying negative pressure (0 to –80 mm Hg) to the membrane caused a reversible, pressure-dependent increase in channel activity in cell-attached and inside-out patches (100 m GTP in bath). Membrane stretch did not alter the sensitivity of the K_ACh channel to GTP. When G _K was maximally activated with 100 m GTPS in inside-out patches, the K_ACh channel activity could be further increased by negative pressure. Trypsin (0.5 mg/ ml) applied to the membrane caused activation of the K_ACh channel in the absence of ACh and GTP; K_ACh channel activity was further increased by stretch. These results indicate that the atrial muscarinic K⁺ channels are modulated by stretch independently of receptor/G protein, probably via a direct effect on the channel protein/ lipid bilayer.