Incorporation of plastoquinone and ubiquinone into liposome membranes studied by HPLC analysis. The effect of side chain length and redox state of quinone

The efficiency of incorporation of plastoquinones and ubiquinones into phospholipid liposomes has been studied. The representatives of short (PQ1 and UQ1) middle (PQ4 and UQ4) and long (PQ9, UQ9 and UQ10) prenylquinones have been used to investigate the effect of quinone side chain length. The properties of hydroquinones have been also thoroughly examined in relation to the quinone forms. The extraction procedure was modified and further developed which enables removing of nonincorporated quinone by pentane washing and then determination of quinone content inside the lipid bilayer. The quantitatively evaluation of the amount of prenylquinone was assayed by means of HPLC analysis which offers much greater sensitivity and could be easily applied in case of hydroquinones. It has been found that PQ1 and UQ1 as well as their reduced forms were present mainly (about 80%) in the aqueous phase, when attempting to introduce them into phospholipid bilayer. In case of quinones having four and more isoprenyl units in side chain, a high level of quinone incorporation, ranging about 95%, was observed. The results pointed out that when comparing the effects of different exogenous quinones on membrane related processes, one has to consider the effectiveness of their incorporation within lipid bilayer.     

Electrochemical measurement of lateral diffusion coefficients of ubiquinones and plastoquinones of various isoprenoid chain lengths incorporated in model bilayers.

The long-range diffusion coefficients of isoprenoid quinones in a model of lipid bilayer were determined by a method avoiding fluorescent probe labeling of the molecules. The quinone electron carriers were incorporated in supported dimyristoylphosphatidylcholine layers at physiological molar fractions (<3 mol%). The elaborate bilayer template contained a built-in gold electrode at which the redox molecules solubilized in the bilayer were reduced or oxidized. The lateral diffusion coefficient of a natural quinone like UQ10 or PQ9 was 2.0 +/- 0.4 x 10(-8) cm2 s(-1) at 30 degrees C, two to three times smaller than the diffusion coefficient of a lipid analog in the same artificial bilayer. The lateral mobilities of the oxidized or reduced forms could be determined separately and were found to be identical in the 4-13 pH range. For a series of isoprenoid quinones, UQ2 or PQ2 to UQ10, the diffusion coefficient exhibited a marked dependence on the length of the isoprenoid chain. The data fit very well the quantitative behavior predicted by a continuum fluid model in which the isoprenoid chains are taken as rigid particles moving in the less viscous part of the bilayer and rubbing against the more viscous layers of lipid heads. The present study supports the concept of a homogeneous pool of quinone located in the less viscous region of the bilayer.     

An electrochemical approach of the redox behavior of water insoluble ubiquinones or plastoquinones incorporated in supported phospholipid layers

Physiological mole fractions of long isoprenic chain ubiquinone (UQ[10]) and plastoquinone (PQ9) were incorporated inside a supported bilayer by vesicle fusion. The template of the bilayer was an especially designed microporous electrode that allows the direct electrochemistry of water insoluble molecules in a water environment. The artificial structure, made by self-assembly procedures, consisted of a bilayer laterally in contact with a built-in gold electrode at which direct electron transfers between the redox heads of the quinones molecules and the electrode can proceed. The mass balances of quinone and lipid in the structure were determined by radiolabeling and spectrophotometry. A dimyristoyl phosphatdylcholine stable surface concentration of 250 +/- 50 pmol x cm(-2), unaffected by the presence of the quinone, was measured in the fluid monolayer. The mole fraction of quinone was between 1 and 3 mol%, remaining unchanged when going from the vesicles to the supported layers. The lipid molecules and the quinone pool were both laterally mobile, and cyclic voltammetry was used to investigate the redox properties of UQ10 and PQ9 over a wide pH range. Below pH 12, the two electrons-two protons electrochemical process at the gold electrode appeared under kinetic control. Thus all thermodynamic deductions must be anchored in the observed reversibility of the quinone/hydroquinol anion transformation at pH > 13. Within the experimental uncertainty, the standard potentials and the pK(a)'s of the pertinent redox forms of UQ10 and PQ9 were found to be essentially identical. This differs slightly from the literature in which the constants were deduced from the studies of model quinones in mixed solvents or of isoprenic quinones without a lipidic environment.     

Physiological links among alternative electron transport pathways that reduce and oxidize plastoquinone in Arabidopsis

In addition to linear electron transport from water to NADP⁺, alternative electron transport pathways are believed to regulate photosynthesis. In the two routes of photosystem I (PSI) cyclic electron transport, electrons are recycled from the stromal reducing pool to plastoquinone (PQ), generating additional ΔpH (proton gradient across thylakoid membranes). Plastid terminal oxidase (PTOX) accepts electrons from PQ and transfers them to oxygen to produce water. Although both electron transport pathways share the PQ pool, it is unclear whether they interact in vivo. To investigate the physiological link between PSI cyclic electron transport‐dependent PQ reduction and PTOX‐dependent PQ oxidation, we characterized mutants defective in both functions. Impairment of PSI cyclic electron transport suppressed leaf variegation in the Arabidopsis immutans (im) mutant, which is defective in PTOX. The im variegation was more effectively suppressed in the pgr5 mutant, which is defective in the main pathway of PSI cyclic electron transport, than in the crr2‐2 mutant, which is defective in the minor pathway. In contrast to this chloroplast development phenotype, the im defect alleviated the growth phenotype of the crr2‐2 pgr5 double mutant. This was accompanied by partial suppression of stromal over‐reduction and restricted linear electron transport. We discuss the function of the alternative electron transport pathways in both chloroplast development and photosynthesis in mature leaves.     

Graviresponse in higher plants and its regulation in molecular bases: relevance to growth and development, and auxin polar transport in etiolated pea seedlings]

We review the graviresponse under true and simulated microgravity conditions on a clinostat in higher plants, and its regulation in molecular bases, especially on the aspect of auxin polar transport in etiolated pea (Pisum sativum L. cv. Alaska) seedlings which were the plant materials subjected to STS-95 space experiments. True and simulated microgravity conditions substantially affected growth and development in etiolated pea seedlings, especially the direction of growth of stems and roots, resulting in automorphosis. In etiolated pea seedlings grown in space, epicotyls were the most oriented toward the direction far from the cotyledons, and roots grew toward the aerial space of Plant Growth Chamber. Automorphosis observed in space were well simulated by a clinorotation on a 3-dimensional clinostat and also phenocopied by the application of auxin polar transport inhibitors of 2,3,5-triiodobenzoic acid, N-(1-naphtyl)phthalamic acid and 9-hydroxyfluorene-9-carboxylic acid. Judging from the results described above together with the fact that activities of auxin polar transport in epicotyls of etiolated pea seedlings grown in space substantially were reduced, auxin polar transport seems to be closely related to automorphosis. Strenuous efforts to learn in molecular levels how gravity contributes to the auxin polar transport in etiolated pea epicotyls resulted in successful identification of PsPIN2 and PsAUX1 genes located in plasma membrane which products are considered to be putative efflux and influx carriers of auxin, respectively. Based on the results of expression of PsPIN2 and PsAUX1 genes under various gravistimulations, a possible role of PsPIN2 and PsAUX1 genes for auxin polar transport in etiolated pea seedlings will be discussed.     

Quinones in long-lived clk-1 mutants of Caenorhabditis elegans

Ubiquinone (UQ) (coenzyme Q) is a lipophilic redox-active molecule that functions as an electron carrier in the mitochondrial electron transport chain. Electron transfer via UQ involves the formation of semiubiquinone radicals, which causes the generation of superoxide radicals upon reaction with oxygen. In the reduced form, UQ functions as a lipid-soluble antioxidant, and protects cells from lipid peroxidation. Thus, UQ is also important as a lipophilic regulator of oxidative stress. Recently, a study on long-lived clk-1 mutants of Caenorhabditis elegans demonstrated that biosynthesis of UQ is dramatically altered in mutant mitochondria. Demethoxy ubiquinone (DMQ), that accumulates in clk-1 mutants in place of UQ, may contribute to the extension of life span. Here we elucidate the possible mechanisms of life span extension in clk-1 mutants, with particular emphasis on the electrochemical property of DMQ. Recent findings on the biochemical function of CLK-1 are also discussed.     

Vesicular and nonvesicular transport of ceramide from ER to the Golgi apparatus in yeast.

Transport and sorting of lipids must occur with specific mechanisms because the membranes of intracellular organelles differ in lipid composition even though most lipid biosynthesis begins in the ER. In yeast, ceramide is synthesized in the ER and transferred to the Golgi apparatus where inositolphosphorylceramide (IPC) is formed. These two facts imply that ceramide can be transported to the Golgi independent of vesicular traffic because IPC synthesis still continues when vesicular transport is blocked in sec mutants. Nonvesicular IPC synthesis in intact cells is not affected by ATP depletion. Using an in vitro assay that reconstitutes the nonvesicular pathway for transport of ceramide, we found that transport is temperature and cytosol dependent but energy independent. Preincubation of ER and Golgi fractions together at 4 degrees C, where ceramide transport does not occur, rendered the transport reaction membrane concentration independent, providing biochemical evidence that ER-Golgi membrane contacts stimulate ceramide transport. A cytosolic protease-sensitive factor is required after establishment of ER-Golgi contacts.     

Low temperature effects on ubiquinone content, respiration rates and lipid peroxidation levels of etiolated seedlings of two differentially chilling-sensitive species

Ubiquinone functions primarily in the electron transport chain of the mitochondria of plants and animals. Secondary roles in plant tissue, such as antioxidant activity, have also been proposed. The effect of low temperature exposure on etiolated seedling embryonic axes of two differentially chilling-sensitive species, mung bean ( Vigna radiata L.) (chilling-sensitive) and pea ( Pisum sativum L. cv. Lincoln) (chilling-tolerant) with respect to respiration rate, lipid peroxidation and ubiquinone content was examined. Whole seedlings (embryonic axis and cotyledon) of both species were exposed to control temperatures (20°C) (6 days) or an acclimatory low temperature treatment of 10°C (3 days) followed by exposure at 5°C (3 days). Measurements were initiated 3 days after seedlings had reached 50% germination (D0). Prior to measurements the cotyledons were removed and only the embryonic axis was used in these experiments. Ubiquinol (UQH₂), ubiquinone (UQ) and total ubiquinone (UQ_tot) content decreased in mung bean in response to the temperature treatment and UQH₂ and UQ_tot remained stable in the more chilling-tolerant pea. The reduction of the total Q-pool was approximately 85–92%, suggesting a high degree of saturation of the respiration pathways. Respiration declined and the RQ ratio increased in both species in response to low temperature. Cytochrome c oxidase (COX) (EC 1.9.3.1) activity was higher in pea than in mung bean but decreased during low temperature exposure in both species. Considering that levels of MDA (lipid peroxidation) did not increase in either species in response to chilling, decreased levels of UQH₂ and UQ observed in chilling-sensitive mung bean may indicate that these compounds were damaged prior to other membrane lipids during low temperature treatment and rendered undetectable.     

Graviresponse and its regulation from the aspect of molecular levels in higher plants: growth and development, and auxin polar transport in etiolated pea seedlings under microgravity.

In STS-95 space experiments we have demonstrated that microgravity conditions resulted in automorphosis in etiolated pea (Pisum sativum L. cv. Alaska) seedlings (Ueda et al. 1999). Automorphosis-like growth and development in etiolated pea seedlings were also induced under simulated microgravity conditions on a 3-dimensional (3-D) clinostat, epicotyls being the most oriented toward the direction far from the cotyledons. Detail analysis of epicotyl bending revealed that within 36 h after watering, no significant difference in growth direction of epicotyls was observed in between seedlings grown on the 3-D clinostat and under 1 g conditions, differential growth near the cotyledonary node resulting in epicotyl bending of ca. 45 degrees toward the direction far from the cotyledons. Thereafter epicotyls continued to grow almost straightly keeping this orientation on the 3-D clinostat. On the other hand, the growth direction in etiolated seedlings changed to antigravity direction by negative gravitropic response under 1 g conditions. Automorphological epicotyl bending was also phenocopied by the application of auxin polar transport inhibitors such as 9-hydroxyfluorene-9-carboxylic acid, N-(1-naphtyl)phthalamic acid and 2,3,5-triiodobenzoic acid. These results together with the fact that auxin polar transport activity in etiolated pea epicotyls was substantially reduced in space suggested that reduced auxin polar transport is closely related to automorphosis. Strenuous efforts to learn how gravity contributes to the auxin polar transport in etiolated pea epicotyls in molecular bases resulted in successful identification of PsPIN2 and PsAUX1 encoding putative auxin-efflux and influx carrier proteins, respectively. Based on the results of these gene expression under simulated microgravity conditions, a possible role of PsPIN2 and PsAUX1 genes for auxin polar transport in etiolated pea seedlings will be discussed.     

Ykt6 forms a SNARE complex with syntaxin 5, GS28, and Bet1 and participates in a late stage in endoplasmic reticulum-Golgi transport

The yeast SNARE Ykt6p has been implicated in several trafficking steps, including vesicular transport from the endoplasmic reticulum (ER) to the Golgi, intra-Golgi transport, and homotypic vacuole fusion. The functional role of its mammalian homologue (Ykt6) has not been established. Using antibodies specific for mammalian Ykt6, it is revealed that it is found mainly in Golgi-enriched membranes. Three SNAREs, syntaxin 5, GS28, and Bet1, are specifically associated with Ykt6 as revealed by co-immunoprecipitation, suggesting that these four SNAREs form a SNARE complex. Double labeling of Ykt6 and the Golgi marker mannosidase II or the ER-Golgi recycling marker KDEL receptor suggests that Ykt6 is primarily associated with the Golgi apparatus. Unlike the KDEL receptor, Ykt6 does not cycle back to the peripheral ER exit sites. Antibodies against Ykt6 inhibit in vitro ER-Golgi transport of vesicular stomatitis virus envelope glycoprotein (VSVG) only when they are added before the EGTA-sensitive stage. ER-Golgi transport of VSVG in vitro is also inhibited by recombinant Ykt6. In the presence of antibodies against Ykt6, VSVG accumulates in peri-Golgi vesicular structures and is prevented from entering the mannosidase II compartment, suggesting that Ykt6 functions at a late stage in ER-Golgi transport. Golgi apparatus marked by mannosidase II is fragmented into vesicular structures in cells microinjected with Ykt6 antibodies. It is concluded that Ykt6 functions in a late step of ER-Golgi transport, and this role may be important for the integrity of the Golgi complex.     

The existence of a lysosomal redox chain and the role of ubiquinone

Several studies concerning the distribution of ubiquinone (UQ) in the cell report a preferential accumulation of this biogenic quinone in mitochondria, plasma membranes, Golgi vesicles, and lysosomes. Except for mitochondria, no recent comprehensive experimental evidence exists on the particular function of UQ in these subcellular organelles. The aim of a recent study was to elucidate whether UQ is an active part of an electron-transfer system in lysosomes. In the present work, a lysosomal fraction was prepared from a light mitochondrial fraction of rat liver by isopycnic centrifugation. The purity of our preparation was verified by estimation of the respective marker enzymes. Analysis of lysosomes for putative redox carriers and redox processes in lysosomes was carried out by optical spectroscopy, HPLC, oxymetry, and ESR techniques. UQ was detected in an amount of 2.2 nmol/mg of protein in lysosomes. Furthermore, a b-type cytochrome and a flavin-adenine dinucleotide (FAD) were identified as other potential electron carriers. Since NADH was reported to serve as a substrate of UQ redox chains in plasma membranes, we also tested this reductant in lysosomes. Our experiments demonstrate a NADH-dependent reduction of UQ by two subsequent one-electron-transfer steps giving rise to the presence of ubisemiquinone and an increase of the ubiquinol pool in lysosomes. Lysosomal NADH oxidation was accompanied by an approximately equimolar oxygen consumption, suggesting that O(2) acts as a terminal acceptor of this redox chain. DMPO/(*)OH spin adducts were detected by ESR in NADH-supplemented lysosomes, suggesting a univalent reduction of oxygen. The kinetic analysis of redox changes in lysosomes revealed that electron carriers operate in the sequence NADH > FAD > cytochrome b > ubiquinone > oxygen. By using the basic spin label TEMPAMINE, we showed that the NADH-related redox chain in lysosomes supports proton accumulation in lysosomes. In contrast to the hypothesis that UQ in lysosomes is simply a waste product of autophagy in the cell, we demonstrated that this lipophilic electron carrier is a native constituent of a lysosomal electron transport chain, which promotes proton translocation across the lysosomal membrane.     

Critical aspects of the antioxidant function of coenzyme Q in biomembranes.

Coenzyme Q (ubiquinone, UQ) is increasingly considered as a significant natural antioxidant, which protects biomembranes in concert with alpha-tocopherol. In vitro experiments demonstrated that reduced UQ (ubiquinol) can improve the chain-breaking activities of alpha-tocopherol by recycling the antioxidant-derived reaction product, the chromanoxyl radical, to the native antioxidant. Less attention, however, was devoted to the antioxidant-derived reaction products of reduced UQ. Although both alpha-tocopherol and ubiquinol were found to be equally effective in scavenging chain-propagating lipid radicals. alpha-tocopherol protected lipid membranes from lipid peroxidation more efficiently than ubiquinol. The present study not only provides data which document this discrepancy but also contributes experimental data on the existence of ubiquinol derived pro-oxidants, which give an explanation of this phenomenon.     

On the participation of chloride in bicarbonate-induced reversal of anion inhibition of photosystem II electron transport in spinach thylakoids

Inhibition of electron transport through photosystem II (PS II) by formate (HCO⁻₂) or nitrite (NO⁻₂) in the presence or absence of chloride ions was studied. The inhibition induced by HCO⁻₂ or NO⁻₂ is overcome by HCO⁻₃ more in the presence, than in the absence of Cl⁻. The data on electron transport are supported by chlorophyll a fluorescence measurements. In experiments. In experiments in which water oxidation was blocked. Cl⁻ was found to facilitate electron transport between bound quinone A (Q⁻_A) and the plastoquinone (PQ) pool. It can thus be concluded that in addition to the well known site of action of Cl⁻ on water oxidation, another site of Cl⁻ action is between Q⁻_A and the PQ pool.     

Characteristics of Cultured Tobacco Cell Strains Producing High Levels of Ubiquinone-10 Selected by a Cell Cloning Technique

Characteristic differences were examined between tobacco cell strains producing high levels of ubiquinone (UQ) and the original tobacco cell line (Nicotiana tabacum L. cv BY-2). The growth rate of strains producing high levels of UQ was about half of that of the original cells. The maximum yield in cell dry weight was about two-thirds of that of original cells. The time-course of UQ formation by selected strains and the respiratory rates were similar to those in the original cells. The UQ contents were much higher than those in original cells, not only per g-dry weight but also per cell. Most UQ in the selected strains were also localized in mitochondria, as well as in the original cells. On protein basis, the yield of purified mitochondria from strains producing high levels of UQ was 4.3 times as much as that from original cells. UQ formation per mg of mitochondrial protein and the molar ratios of UQ to the other electron transport components in selected cells were similar to those in original cells. The ratio of the mitochondrial protein yield in strains producing high levels of UQ to the yield in original cells correlated closely with the ratio of UQ content per g-dry weight in UQ-producing strains to UQ content in original cells.     

Effects of UV-B radiation on Chl fluorescence of greening barley (Hordeum vulgare L.) seedling

Effects of UV-B radiation on the developing chloroplast of barley (Hordeum vulgare L.) seedling during greening were determined by Chl contents, Fo, Fv and fluorescence quenching coefficients. In greening of etiolated barley seedling, the value of Fo was greatly increased after the initiation of greening. However Fv and Fv/Fo were gradually increased. In greening with the additional irradiation of UV-B radiation, the value of Fo was strikingly decreased than that of the control after the initiation of greening, but Fv was gradually decreased from than that of the control during the greening period. These results suggest that the function of light-harvesting Chl are more sensitive than photosynthetic electron transport system by UV-B. Chl contents, Fv/Fo, qP and qNP, were decreased from than that of the control during the 72 h greening, especially, qR was strikingly decreased, but qE was slightly decreased by UV-B. These suggest that the sites of inhibition by UV-B are PSII and all sites of photosynthetic electron transport system. But PQ pool seems to be slightly inhibited by UV-B.     

Gel-phase microdomains and lipid rafts in monolayers affect the redox properties of ubiquinone-10

The redox properties of ubiquinone-10 (UQ) were examined in monolayers of mixtures of dioleoylphosphatidylcholine, palmitoylsphingomyelin, and cholesterol of different compositions, self-assembled on a mercury electrode, over the pH range from 7.5 to 9.5. A detailed analysis of the cyclic voltammograms of UQ in the above lipid environments points to a mechanism consisting of an elementary electron transfer step followed by two protonation (or deprotonation) steps in quasiequilibrium and by a further electron transfer step. In a lipid environment of solid-ordered (s_o) microdomains in a liquid-disordered (l_d) matrix, electron transport across the lipid monolayer takes place in the l_d phase. In a pure s_o phase, UQ tends to segregate into UQ-rich pools, exhibiting reversible electron transfer steps. In a lipid environment consisting of liquid-ordered (l_o) microdomains (lipid rafts) in an l_d matrix, UQ molecules tend to localize along the edge of the lipid rafts. However, in a lipid environment consisting exclusively of l_o and s_o microdomains, UQ molecules tend to segregate into UQ-rich pools. In all lipid environments, electron transport by UQ occurs with the quinone moiety localized on the solution side with respect to the ester linkages of the dioleoylphosphatidylcholine molecules.     

Paraquat toxicity is reduced by polyamines in rice leaves

The protective effect of polyamines against paraquat (PQ) toxicity of rice (Oryza sativa) leaves was investigated. PQ treatment resulted in a higher putrescine (PUT) and lower spermidine (SPD) and spermine (SPM) levels in rice leaves. Pretreatment with SPD and SPM, which resulted in a 10- and 20-fold increase in endogenous level of SPD and SPM, respectively, reduced PQ toxicity (30%). Limited reduction of PQ toxicity by exogenous SPD and SPM is most likely due to the fact that they are not readily transported in rice leaf cells and localized to those areas along the cut edges of detached rice leaves [4]. PUT pretreatment did not increase endogenous SPD and SPM levels and had no effect on reducing PQ toxicity. It was found that 1,10-phenanthroline, an iron chelator, treatment reduced the toxicity of PQ (35%) and increased the levels of SPD (27%). The results indicate that reduction of PQ toxicity by SPD and SPM is due to increased activities of catalase (18%) and peroxidase (40%).     

Changes in Mitochondrial Respiratory Chain Components of Petunia Cells during Culture in the Presence of Antimycin A

When petunia (Petunia hybrida Vilm, cv Rosy Morn) cells are cultured in the presence of 2 [mu]M antimycin A (AA), respiration proceeds mainly via the cyanide-resistant pathway. Cyanide-resistant respiratory rates were higher in mitochondria from AA cells than in control mitochondria. Compared with control cells, an increase in alternative oxidase protein was observed in AA cells, as well as an increase in ubiquinone (UQ) content. A change in the kinetics of succinate dehydrogenase was observed: there was a much higher activity at high UQ reduction in mitochondria from AA cells compared with control mitochondria. No changes were found for external NADH dehydrogenase kinetics. In AA cells in vivo, UQ reduction was only slightly higher than in control cells, indicating that increased electron transport via the alternative pathway can prevent high UQ reduction levels. Moreover, O2 consumption continues at a similar rate as in control cells, preventing O2 danger. These adaptations to stress conditions, in which the cytochrome pathway is restricted, apparently require, in addition to an increase in alternative oxidase protein, a new setup of the relative amounts and/or kinetic parameters of all of the separate components of the respiratory network.     

Structure and properties of a dimeric N-terminal fragment of human ubiquitin.

Previous peptide dissection and kinetic experiments have indicated that in vitro folding of ubiquitin may proceed via transient species in which native-like structure has been acquired in the first 45 residues. A peptide fragment, UQ(1-51), encompassing residues 1 to 51 of ubiquitin was produced in order to test whether this portion has propensity for independent self-assembly. Surprisingly, the construct formed a folded symmetrical dimer that was stabilised by 0.8 M sodium sulphate at 298 K (the S state). The solution structure of the UQ(1-51) dimer was determined by multinuclear NMR spectroscopy. Each subunit of UQ(1-51) consists of an N-terminal beta-hairpin followed by an alpha-helix and a final beta-strand, with orientations similar to intact ubiquitin. The dimer is formed by the third beta-strand of one subunit interleaving between the hairpin and third strand of the other to give a six-stranded beta-sheet, with the two alpha-helices sitting on top. The helix-helix and strand portions of the dimer interface also mimic related features in the structure of ubiquitin. The structural specificity of the UQ(1-51) peptide is tuneable: as the concentration of sodium sulphate is decreased, near-native alternative conformations are populated in slow chemical exchange. Magnetization transfer experiments were performed to characterize the various species present in 0.35 M sodium sulphate, namely the S state and two minor forms. Chemical shift differences suggest that one minor form is very similar to the S state, while the other experiences a significant conformational change in the third strand. A segmental rearrangement of the third strand in one subunit of the S state would render the dimer asymmetric, accounting for most of our results. Similar small-scale transitions in proteins are often invoked to explain solvent exchange at backbone amide proton sites that have an intermediate level of protection.     

Properties of a GTP sensitive microdomain in rough microsomes

Stripped rough microsomes (SRM) fuse when incubated with physiological concentrations of GTP and MgCl2. In order to examine further to what extent such fusions are associated with other membrane functions of rough endoplasmic reticulum, we have evaluated the role of cytosolically exposed peptide constituents of SRM in fusion, and the possible relationship of GTP/MgCl2-induced fusion in protein transport across endoplasmic reticulum (ER) membranes, and in ER-Golgi interactions. Controlled proteolytic digestion of SRM led to the loss of fusion capability at 15 micrograms/ml trypsin--a concentration which maintained the latency of intraluminal mannose-6-phosphatase. Hence, a cytosolically exposed protein(s) regulated fusion. Based on ribonuclease-induced ribosome capping experiments, it was further concluded that the cytosolic oriented protein(s) was sequestered beneath the ribosome. As co-translational cell free translocation of placental lactogen across SRM was similar in control membranes compared to those rendered incapable of fusing, it was concluded that the fusion phenomenon may not be related to translocation. Under conditions promoting homologous fusion of SRM or Golgi membranes, mixtures of the two membranes showed no heterologous membrane fusion as assessed morphologically or by the transport of newly synthesized membrane glycoprotein. These experiments attest to the specificity of cytosolically exposed protein(s) in regulating nucleotide/divalent cation-induced membrane fusion.