Different routes for integral protein insertion into Ricinus communis protein-body and glyoxysome membranes

Total polyadenylated RNA from ripening or germinating Ricinus communis L. endosperm was translated in rabbit reticulocyte lysate in the absence or presence of canine pancreatic microsomes. The products were immunoprecipitated using antibodies raised againts Triton X-114-extracted integral membrane proteins of protein bodies or glyoxysomes. While the proteins of proteinbody membranes were found to insert co-translationally into added microsomes, this was not observed in the case of glyoxysomal proteins. This observation was confirmed using antibodies raised against a purified glyoxysome membrane protein, alkaline lipase. These results indicate that different routes exist for the insertion of membrane proteins into the two organelles. In both cases membrane-protein insertion does not appear to be accompanied by proteolytic processing.Abbreviations anti-PB antiserum to integral protein-body membrane proteins - anti-G antiserum to integral glyoxysomal membrane proteins - anti-L antiserum to alkaline lipase - ER endoplasmic reticulum - M_r relative molecular mass - mRNA poly(A)-rich messenger RNA - PAGE polyacrylamide gel electrophoresis - poly(A) polyadenylic acid - SDS sodium dodecyl sulphate     

Nitrite reduction and carbohydrate metabolism in plastids purified from roots of Pisum sativum L.

Nitrate reduction in roots and shoots and exchange of reduced N between organs were quantitatively estimated in intact 13-d-old seedlings of two-row barley (Hordeum vulgare L. cv. Daisengold) using the ¹⁵N-incorporation model (A. Gojon et al. (1986) Plant Physiol. 82, 254–260), except that NH ₊ ⁴ was replaced by NO _- ² . N-depleted seedlings were exposed to media containing both nitrate (1.8 mM) and nitrite (0.2 mM) under a light-dark cycle of 12:12 h at 20°C; the media contained different amounts of ¹⁵N labeling. Experiments were started either immediately after the beginning (expt. 1) or immediately prior to the end (expt. 2) of the light period, and plants were sampled subsequently at each light-dark transition throughout 36 h. The plants effectively utilized ¹⁵NO _- ³ and accumulated it as reduced ¹⁵N, predominantly in the shoots. Accumulation of reduced ¹⁵N in both experiments was nearly the same at the end of the experiment but the accumulation pattern in roots and shoots during each 12-h period differed greatly depending on time and the light conditions. In expt. 1, the roots accounted for 31% (light), 58% (dark), and 9% (light) of nitrate reduction by the whole plants, while in expt. 2 the contributions of the root were 82% (dark), 20% (light), and 29% (dark), during each of the three 12-h periods. Xylem transport of nitrate drastically decreased in the dark, but that of reduced N rather increased. The downward translocation of reduced ¹⁵N increased while nitrate reduction in the root decreased, whereas upward translocation decreased while nitrate reduction in the shoot increased. We conclude that the cycling of reduced N through the plant is important for N feeding of each organ, and that the transport system of reduced N by way of xylem and phloem, as well as nitrate reduction by root and shoot, can be modulated in response to the relative magnitude of reduced-N demands by the root and shoot, with the one or the other predominating under different circumstances.Symbols Anl accumulation of reduced ¹⁵N from ¹⁵NO _- ³ in ¹⁴NO _- ³ -fed roots of divided root system - Ar accumulation in root of reduced ¹⁵N from ¹⁵NO _- ³ - As accumulation in shoot of reduced ¹⁵N from ¹⁵NO _- ³ - Rr ¹⁵NO _- ³ reduction in root - Rs ¹⁵NO _- ³ reduction in shoot - Tp translocation to root of shoot-reduced ¹⁵N from ¹⁵NO _- ³ in phloem - Tx translocation to shoot of root-reduced ¹⁵N from ¹⁵NO _- ³ in xylem     

Selective illumination of single photoreceptors in the house fly retina: local membrane turnover and uptake of extracellular horseradish peroxidase (HRP) and Lucifer Yellow

Summary Single photoreceptor cells in the compound eye of the housefly Musca domestica were selectively illuminated and subsequently compared electron-microscopically with the unilluminated photoreceptors in the immediate surroundings. The rhabdomeres of the illuminated cells remain largely unaffected, but the cells show an increase in the number of coated pits, various types of vesicles, and degradative organelles; some of the latter organelles are described for the first time in fly photoreceptors. Coated pits are found not only at the bases of the microvilli, but also in other parts of the plasma membrane. Degradative organelles, endoplasmic reticulum (ER) and mitochondria aggregate in the perinuclear region. The rough ER and smooth ER are more elaborate, the number of Golgi stacks, free ribosomes and polysomes is increased, and the shape and distribution of heterochromatin within the nuclei are altered. Illuminated photoreceptors also interdigitate extensively with their neighbouring secondary pigment cells. These structural changes in illuminated fly photoreceptor cells indicate an increase in membrane turnover and cellular metabolism. When applied to the eye, Lucifer Yellow spreads into the extracellular space and is taken up only by the illuminated photoreceptor cells. These cells show the same structural modifications as above. Horseradish peroxidase applied in the same way is observed in pinocytotic vesicles and degradative organelles of the illuminated cells. Hence, the light-induced uptake of extracellular compounds takes place in vivo at least partially as a result of an increase in pinocytosis.     

A marker of acid-secreting membrane movement in rat gastric parietal cells

A monoclonal antibody (mab 146.14) marker of the movement of acid-secreting membranes in rat gastric parital cells has been produced and characterized. Mab 146.14 recognized a 95-kD major component of a purified membrane fraction of rat gastric mucosa, the protein composition of which was similar to that of well characterized porcine H+ -K+ ATPase-enriched membranes, and that presented the characteristic shift of density depending on whether it was purified from resting or stimulated tissues. Further biochemical analysis characterized the antigen as a membranous protein that might be in its native form, part of a higher multimolecular complex. Immunocytochemical localization of the antigen demonstrated that only membranes related to acid secretion in parietal cells expressed the 95-kD antigen. In resting conditions, the 95-kD antigen was diffusely distributed in the cell cytoplasm associated with inactive tubulovesicles. In stimulated cells, by contrast, all the antigen was recovered associated with secretory active microvilli formed by the apical insertion of the previously resting internal tubulovesicles. In conclusion, the 95-kD antigen, presumably a part of the rat gastric proton pump, is a marker of acid-secreting membranes in rat parietal cells. The translocation of antigen and membranes, observed by both light and electron microscopy supports the fusion model of membrane insertion from a cytoplasmic storage pool to the apical surface upon stimulation of acid secretion.     

Translocation and proteolytic processing of nascent secretory polypeptide chains: two functions associated with the ribosomal domain of the endoplasmic reticulum

Rat liver microsomes were subfractionated by isopycnic centrifugation in sucrose gradient. The subfractions were assayed for translocation and proteolytic processing of nascent polypeptides in a rabbit reticulocyte lysate programmed with total RNA from human term placenta. The distribution of the translocation and processing of prelactogen through the gradient correlated with that of the microsomal RNA (ribosomes). Microsomes became inactive upon incubation with elastase, but the proteolyzed membranes recovered their activity by recombination with the soluble and active fragment of the docking protein (SRP-receptor) from dog pancreas. When this fragment was combined with the gradient subfractions, or with the subfractions inactivated by incubation with elastase, the density profile of the translocation activity remained similar to that of RNA. Thus, its distribution cannot be accounted for merely by that of the docking protein; another membrane constituent, still unidentified, is both necessary for translocation of polypeptides and restricted to the rough portions of the endosplamic reticulum. Signal peptidase was assayed in the absence of protein synthesis, by use of preformed prelactogen and detergent-disrupted microsomes. Its density distribution was also similar to that of RNA. Several components of the endosplamic reticulum now appear to be segregated within restricted areas on either side of the membrane, and to make up a biochemically distinct domain. We propose to call it the ribosomal domain in consideration of its contribution to protein biosynthesis by bound ribosomes. This domain probably accounts for a greater part of the membrane area at the cytoplasmic than at the luminal surface, as postulated earlier to explain how enzymes of the cytoplasmic surface are relatively less abundant in the rough microsomes than those of the luminal surface [Amar-Costesec A. & Beaufay H. (1981) J. Theor. Biol. 89, 217-230].     

普通小麦栽培品种丰抗13的4B—1D染色体易位的鉴定

通过细胞学观察,在普通小麦栽培品种“丰抗13”和“京红1号”的杂交后代中,发现有多价体出现,这就表明有染色体易位发生。为进一步弄清究竟是哪条染色体发生了易位,我们采用单体测交方法,观察鉴定所有各单体系F_1的花粉母细胞第一次减数分裂中期Ⅰ(以下简称PMCs中Ⅰ)染色体构型。从鉴定结果发现,凡2n=42的F_1 PMCs中Ⅰ出现19~Ⅱ 1~Ⅳ,而2n=41的F_1PMCs中Ⅰ的染色体构型不同,单体与易位有关的两个单体系4B和1D F_1 PMCs中的Ⅰ构型中有部分呈现为19个二价体加1个三价体,即19~Ⅱ 1~Ⅲ,没有单价体,而其余各单体系F_1 PMCs中Ⅰ构型则表现为18个二价体,1个四价体和1个单价体,即18~Ⅱ 1~Ⅰ 1~Ⅳ。因此,可以肯定“丰抗13”存在1个染色体易位,其有关染色体就是4B和1D。     

Identification of thiol groups and a disulfide crosslink site in bovine myelin proteolipid protein

The existence of disulfide crosslinks limits the number of possible folded structures a protein can assume. Thus localization of disulfide and thiol groups is a key to understanding the conformation and orientation of myelin proteolipid protein (PLP) in the myelin membrane. [¹⁴C]Carboxamidomethylated PLP was fragmented with chymotrypsin, and the resulting mixture was partially separated by reversed-phase HPLC. Purified ¹⁴C-labeled peptides and a disulfide containing peptide were characterized by amino acid analysis. These experiments showed that Cys-32 and Cys-34 are free thiols, and are presumably on the interior of the cell or within the membrane bilayer, and that Cys-200 and Cys-219 are joined by a disulfide bond, and are probably located on the extracellular face of the membrane. Sequence analysis experiments indicate that Cys-5, Cys-6 and Cys-9 are linked by disulfides, probably to other parts of the protein on the extracellular face of the membrane.     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or chaning the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5′-(N-ethylcar☐amido)adenosine (NECA, 10 μM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 ± 0.02 (S.E.) to 1.80 ± 0.02 (p < 0.001) while inosine (10 μM), a poor adenosine receptor agonist, had no effect (1.73 ± 0.04, p =n.s. vs. control, p < 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA > adenosine = N⁶-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 μM) did not significantly change the number or affinity of [³H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Impact of salt adaptation on esterified fatty acids and cytochrome oxidase in plasma and thylakoid membranes from the cyanobacterium Anacystis nidulans

During adaptation of photoautotrophically growing fresh water cyanobacterium Anacystis nidulans to high salinity the cells showed a pronounced increase of proton-sodium antiporter activity, and of cytochrome c oxidase in isolated and purified plasma membrane. At the same time the concentrations of plasma membrane-bound EDTA-resistant copper and iron (determined by inductively coupled plasma atomic emission spectrometry) rose proportionately, accompanied by an increase in whole cell respiration. In plasma membranes from salt adapted cells lipid/protein ratios were markedly higher than in control cells, levels of esterified saturated and long-chain fatty acids being significantly higher than the respective levels of unsaturated and short-chain fatty acids which explains the higher lipid-phase transition temperatures derived from Arrhenius plots. Immunoblotting of the membrane proteins with antisera raised against the cytochrome c oxidases from Paracoccus denitrificans and A. nidulans gave two cross-reacting bands with apparent molecular weights around 50000 and 30000 (subunits I and II, respectively) which were more pronounced in plasma membranes from salt adapted cells when compared to control cells. The protein pattern of plasma membranes from salt adapted cells also showed the appearance of bands at apparent molecular weights of 44000–48000 and 54000–56000 which might stem from the proton/sodium-antiporter in this membrane.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - cyt cytochrome - DCCD N,N-dicyclohexylcarbodiimide - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - ICP-AES inductively coupled plasma atomic emission spectrometry - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - EPR electron paramagnetic resonance spectrometry     

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains. The enzyme in strain BK5 is shown to be both functionally and structurally related to the nitrate reductase of Paracoccus denitrificans and Escherichia coli.Abbreviation TMAO trimethylamine-N-oxide