Endoplasmic Reticulum Membrane Fractions Research Articles

Mitochondrial membrane protein Sigmar1 regulates mitochondrial dynamics and function

Rationale: Sigma 1 receptor (Sigmar1) is a molecular chaperone protein known to reside at the interface between the mitochondria and endoplasmic reticulum (ER) in different cellular systems. We recently reported that Sigmar1 is highly expressed in cardiomyocytes but its molecular function still remains unknown. Objective: We investigated the subcellular localization and molecular function of Sigmar1 in regulating mitochondrial function in the heart. Methods and Results: Subcellular fractionation and biochemical experiments confirmed Sigmar1 expression in the mitochondrial- and mitochondrial-associated ER membrane fraction. We isolated mitochondria from the mouse and human heart cell lysates and found Sigmar1 as an integral mitochondrial outer membrane protein by biochemical experiments. To define the molecular function of Sigmar1, we used the Sigmar1 knockout mice. Sigmar1 knockout mice showed normal morphometry but exhibited significantly altered cardiac hemodynamics and ultrastructure. Ultrastructural analysis by transmission electron microscopy revealed accumulation of irregular shaped highly fused mitochondria and sarcomere disorganization. Examining the mitochondrial dynamics regulatory proteins, we found that Sigmar1 KO hearts showed decreased level of the fission protein, Drp-1 and increased expression of fusion protein, OPA1. Analysis of mitochondrial bioenergetics showed significantly reduced basal respiration and ATP turnover in the mitochondria isolated from Sigmar1 KO hearts. We also found decreased mitochondrial DNA copy number and ATP content in the Sigmar1 KO hearts. Conclusions: Our findings suggested that Sigmar1 is an integral mitochondrial outer membrane protein necessary to maintain normal mitochondrial morphology and function in the heart.

Perilipin-2 Deletion Impairs Hepatic Lipid Accumulation by Interfering with Sterol Regulatory Element-binding Protein (SREBP) Activation and Altering the Hepatic Lipidome

Perilipin-2 (PLIN2) is a constitutively associated cytoplasmic lipid droplet coat protein that has been implicated in fatty liver formation in non-alcoholic fatty liver disease. Mice with or without whole-body deletion of perilipin-2 (Plin2-null) were fed either Western or control diets for 30 weeks. Perilipin-2 deletion prevents obesity and insulin resistance in Western diet-fed mice and dramatically reduces hepatic triglyceride and cholesterol levels in mice fed Western or control diets. Gene and protein expression studies reveal that PLIN2 deletion suppressed SREBP-1 and SREBP-2 target genes involved in de novo lipogenesis and cholesterol biosynthetic pathways in livers of mice on either diet. GC-MS lipidomics demonstrate that this reduction correlated with profound alterations in the hepatic lipidome with significant reductions in both desaturation and elongation of hepatic neutral lipid species. To examine the possibility that lipidomic actions of PLIN2 deletion contribute to suppression of SREBP activation, we isolated endoplasmic reticulum membrane fractions from long-term Western diet-fed wild type (WT) and Plin2-null mice. Lipidomic analyses reveal that endoplasmic reticulum membranes from Plin2-null mice are markedly enriched in ω-3 and ω-6 long-chain polyunsaturated fatty acids, which others have shown inhibit SREBP activation and de novo lipogenesis. Our results identify PLIN2 as a determinant of global changes in the hepatic lipidome and suggest the hypothesis that these actions contribute to SREBP-regulated de novo lipogenesis involved in non-alcoholic fatty liver disease.

Autophagosomes form at ER–mitochondria contact sites

Autophagy is a tightly regulated intracellular bulk degradation/recycling system that has fundamental roles in cellular homeostasis. Autophagy is initiated by isolation membranes, which form and elongate as they engulf portions of the cytoplasm and organelles. Eventually isolation membranes close to form double membrane-bound autophagosomes and fuse with lysosomes to degrade their contents. The physiological role of autophagy has been determined since its discovery, but the origin of autophagosomal membranes has remained unclear. At present, there is much controversy about the organelle from which the membranes originate--the endoplasmic reticulum (ER), mitochondria and plasma membrane. Here we show that autophagosomes form at the ER-mitochondria contact site in mammalian cells. Imaging data reveal that the pre-autophagosome/autophagosome marker ATG14 (also known as ATG14L) relocalizes to the ER-mitochondria contact site after starvation, and the autophagosome-formation marker ATG5 also localizes at the site until formation is complete. Subcellular fractionation showed that ATG14 co-fractionates in the mitochondria-associated ER membrane fraction under starvation conditions. Disruption of the ER-mitochondria contact site prevents the formation of ATG14 puncta. The ER-resident SNARE protein syntaxin 17 (STX17) binds ATG14 and recruits it to the ER-mitochondria contact site. These results provide new insight into organelle biogenesis by demonstrating that the ER-mitochondria contact site is important in autophagosome formation.

Svp26 Facilitates Endoplasmic Reticulum to Golgi Transport of a Set of Mannosyltransferases in Saccharomyces cerevisiae

Svp26 is a polytopic integral membrane protein found in the ER and early Golgi compartment. In the Deltasvp26 cell, the Golgi mannosyltransferase Ktr3 remains in the ER. Here, we report that two other Golgi mannosyltransferases, Mnn2 and Mnn5 are also mislocalized and found in the ER in the absence of Svp26 and that localization of other mannosyltransferases including Mnn1 are not affected. Mnn2 and Mnn5 bind to Svp26 in vivo as Ktr3 does. Using an in vitro budding assay, the incorporation of Ktr3 and Mnn2 in the COPII vesicles is greatly stimulated by the presence of Svp26. As Svp26 itself is an efficient cargo, Svp26 is likely to support selective incorporation of a set of mannosyltransferases into COPII vesicles by working as their adaptor protein. The domain switching between Svp26-dependent Mnn2 or Ktr3 and Svp26-independent Mnn1 suggests that the lumenal domain of mannosyltransferases, but not the cytoplasmic or transmembrane domain, is responsible for recognition by Svp26.

Differences in membrane association and sub-cellular distribution between NS2-3 and NS3 of bovine viral diarrhoea virus

The sub-cellular location and mechanism of membrane association of NS3 and NS2-3 polypeptides of bovine viral diarrhoea virus (BVDV) have been examined. Both NS3 and NS2-3 proteins were detected in post-nuclear membrane fractions but not in cytosolic fractions of BVDV infected cells; a proportion of NS3, but not NS2-3, could be dissociated from the membranes with 800 mM KCl or at pH 11. Following extraction with 1% Triton X-114, NS3 was predominantly present in the aqueous phase, but NS2-3 was only recovered in the detergent phase. Confocal microscopy showed that in BVDV infected cells, NS3 and/or NS2-3 co-localise with the endoplasmic reticulum (ER) protein, ERP60, but not Golgi or lysosomal proteins. Sub-cellular fractionation analysis demonstrated that NS2-3 was almost exclusively associated with the rough ER membrane but a significant proportion of NS3 was present in the smooth ER membrane fractions in addition to the rough ER membrane. These differences in the distribution of NS2-3 and NS3 on ER membranes in cells infected with cytopathogenic (CP) strains of BVDV were also observed using confocal microscopy and antibodies that are specific to either NS2 or NS3. This distinct distribution of NS3 and NS2-3 on the ER membrane has revealed a further difference between CP and non-cytopathogenic (NCP) strains of BVDV.

Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus.

The targeting route of newly synthesized GM130 and GRASP65 to the Golgi apparatus was investigated by three different approaches. First, localization of pulse labeled GM130 and GRASP65 in normal rat kidney (NRK) cells was traced by subcellular fractionation followed by immunoprecipitation. Immediately after the pulse labeling, GM130 and GRASP65 were found in the Golgi but not in the endoplasmic reticulum (ER) membrane fractions, whereas a control Golgi membrane protein was still found in the ER membrane fractions. Second, epitope tagged GM130 and GRASP65 were expressed in NRK cells by plasmid microinjection into the nuclei and their localization was analyzed by immunofluorescence. When ER to Golgi transport was inhibited by prior microinjection of a GTP-restricted mutant of Sar1 protein into the cytosol, the expressed GM130 and GRASP65 showed clear Golgi localization. Last, binding of GM130 and GRASP65 to the membranes was analyzed in vitro. In vitro synthesized GM130 and GRASP65 specifically bound to purified Golgi membranes but not to microsomal membranes. The bound GM130 and GRASP65 were found to form a complex with pre-existing counterparts on the Golgi membrane. These results strongly suggested that GM130 and GRASP65 are directly targeted to the Golgi membrane without initial assembly on the ER and subsequent vesicular transport to the Golgi apparatus.

One of the origins of plasma membrane phosphatidylserine in plant cells is a local synthesis by a serine exchange activity

In plant cells, as in animal cells, the endoplasmic reticulum (ER) is considered to be the major site of phospholipid synthesis, and it has been shown that phosphatidylserine (PS) reaches the plasma membrane via the vesicular ER-Golgi-plasma membrane pathway in leek cells. However, it has never been determined whether the plasma membrane of leek cells is able to synthesize PS. We have analyzed the distribution of PS synthesizing enzymes along the vesicular pathway. In ER, Golgi and plasma membrane fractions isolated from leek cells, we have measured the activity of the two biosynthetic pathways leading to the synthesis of PS, i.e. serine exchange and CTP cytidylyltransferase plus PS synthase. We have found a high serine exchange activity in the plasma membrane fraction, and then determined that this membrane is able to synthesize both long chain fatty acid- and very long chain fatty acid-containing PS. Therefore, the PS in the plasma membrane of leek cells has two different origins: the intracellular vesicular pathway from the ER and a local synthesis in the plasma membrane.

Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure.

Production of extracellular proteins plays an important role in the physiology of Trichoderma reesei and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in T. reesei the DPM1 gene of Saccharomyces cerevisiae, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated DPM1, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript of cbh1, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants, and no hyperglycosylation was detected. Electron microscopy of the DPM1-amplified strains revealed amorphous structure of the cell wall and over three times as many mitochondria as in the control. Our data indicate that molecular manipulation of glycan biosynthesis in Trichoderma can result in improved protein secretion.

Endoplasmic reticulum stress-inducible protein GRP94 is associated with an Mg2+-dependent serine kinase activity modulated by Ca2+ and GRP78/BiP.

The 94-kDa glucose-regulated protein (GRP94) is a glycoprotein in the endoplasmic reticulum (ER). It has been characterized as a Ca2+-binding protein and a molecular chaperone. In this report we show that highly purified GRP94 exhibits an active Mg2+-dependent serine kinase activity (termed 94-kinase). The 94-kinase can be recovered from ER membrane fractions and is able to phosphorylate both the constitutive and stress-induced forms of GRP94, correlating with their induction kinetics. The 94-kinase activity is distinct from casein kinase II. In contrast to the heat-stable, Ca2+-dependent autophosphorylation activity recently reported for GRP94, the labile 94-kinase activity is inhibited by Ca2+. We determined that the phosphopeptide map of in vitro phosphorylated GRP94 by the 94-kinase resembles that of the in vivo phosphorylated GRP94. Further, the 94-kinase activity can be specifically stimulated by GRP78, a coregulated protein in the ER known to interact with GRP94.

Endoplasmic reticulum stress‐inducible protein GRP94 is associated with an Mg2‐dependent serine kinase activity modulated by Ca2 and GRP78/BiP

The 94-kDa glucose-regulated protein (GRP94) is a glycoprotein in the endoplasmic reticulum (ER). It has been characterized as a Ca2+-binding protein and a molecular chaperone. In this report we show that highly purified GRP94 exhibits an active Mg2+-dependent serine kinase activity (termed 94-kinase). The 94-kinase can be recovered from ER membrane fractions and is able to phosphorylate both the constitutive and stress-induced forms of GRP94, correlating with their induction kinetics. The 94-kinase activity is distinct from casein kinase II. In contrast to the heat-stable, Ca2+-dependent autophosphorylation activity recently reported for GRP94, the labile 94-kinase activity is inhibited by Ca2+. We determined that the phosphopeptide map of in vitro phosphorylated GRP94 by the 94-kinase resembles that of the in vivo phosphorylated GRP94. Further, the 94-kinase activity can be specifically stimulated by GRP78, a coregulated protein in the ER known to interact with GRP94. J. Cell. Physiol. 170:115–129, 1997. © 1997 Wiley-Liss, Inc.

The Sec13p complex and reconstitution of vesicle budding from the ER with purified cytosolic proteins.

SEC13 encodes a 33 kDa protein that participates in vesicle budding from the endoplasmic reticulum (ER). In order to purify a functional form of Sec13p, a SEC13-dihydrofolate reductase (mouse) fusion gene (SEC13:DHFR) was constructed that complements both sec13 temperature sensitive and null mutations. Methotrexate-agarose affinity chromatography facilitated the purification of two forms of the Sec13-dhfrp fusion protein: a monomeric form and a high molecular weight complex. The complex form consists of two subunits: Sec13-dhfrp and a 150 kDa protein (p150). Native immunoprecipitation experiments confirm that Sec13p exists in a complex with p150 in wild type cells. Functional analysis supports a role for both subunits in protein transport. Vesicle budding from the ER in a cell-free reaction is inhibited by Fab antibody fragments directed against either Sec13p or p150. The purified Sec13-dhfrp/p150 complex, but not the Sec13-dhfrp monomer, in combination with two other pure protein fractions (Sar1p and a Sec23/Sec24 protein complex) satisfies the requirement for cytosol in a cell-free vesicle budding reaction. The vesicles formed with the purified protein fractions are competent to fuse with the Golgi and are biochemically distinct from the ER membrane fraction from which they derive.

Hormonal regulation of Ca2+ transport in the endomembrane system of the barley aleurone

Gibberellic acid (GA3) and abscisic acid (ABA) regulate α-amylase production in aleurone tissue of the barley (Hordeum vulgare L.) grain during germination. Since α-amylase is a Ca2+-containing protein that is synthesized on the rough endoplasmic reticulum (ER), we investigated the possibility that GA3 and ABA regulate the flux of Ca2+ into the ER of aleurone cells. Membrane vesicles were isolated from GA3- and ABA-treated aleurone layers to characterize the effect of these hormones on calcium transport (CaT) activity as measured by the uptake of 45CaCl2 in vitro. We found that ABA and GA3 greatly alter the relative amounts of CaT activity in ER and tonoplast (TP) membrane fractions. Treatment with GA3 results in CaT being associated primarily with the ER, whereas treatment with ABA results in roughly equal amounts of CaT in ER and TP. Moreover, we found that ABA prevents or reverses the GA3-induced increase in CaT in the ER. The effects of ABA and GA3 on CaT in the ER require several hours to develop, and the effects of these hormones on CaT cannot be brought about by adding the hormones to membranes in vitro. In order to determine whether the in-vitro measurements of CaT reflect changes that occur in vivo we measured the Ca2+ content of isolated ER, measured Ca2+ transport into aleurone layers, and examined differences in the ability of ER proteins from GA- and ABA-treated layers to bind Ca2+. Our results indicate that GA3 increases and ABA decreases both Ca2+ flux into the ER and the amount of calcium that accumulates in the ER of barley aleurone cells in vivo.

The effect of a phorbol ester on the lipid microviscosity of two endoplasmic reticulum membrane fractions isolated from krebs II ascites cells

This paper deals with microviscosity parameters and thermoinduced structural transitions in the lipids of smooth and heavy rough endoplasmic reticulum membranes isolated from Krebs II ascites cells incubated with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate. The phorbol ester was found to bring about a threefold increase in the microviscosity of the lipids in heavy rough membranes. Spin probe I (2,2,6,6-tetrahydro-4-capryloyl-oxypiperidine-1-oxyl), localized in the surface layer of the membrane lipids, gave results which indicate an increased number of thermoinduced structural transitions in the smooth membranes in the treated cells due to the transitions occurring at relatively low temperature and a decreased number of such transitions in the heavy rough fraction especially at high temperature. For 5,6-benzo-2,2,4,4-tetramethyl-1,2,3,4-tetrahydro-gamma-carboline-oxyl, probe II, mainly distributed in the annular lipids, a decrease in the number of low temperature transitions in the smooth fraction was observed, while an increase occurred in the heavy rough one. The results obtained are discussed in terms of the effect of phorbol esters as promoters of tumor progression.

Subcellular localization and hormone sensitivity of adipocyte cyclic AMP phosphodiesterase

Treatment of intact adipocytes with either or both insulin and adrenaline stimulated membrane cyclic AMP phosphodiesterase activity only in the endoplasmic reticulum subfraction. The cyclic GMP-inhibited cyclic AMP phosphodiesterase activity was also found in this fraction. Quantitative Western blotting using a specific polyclonal antibody, raised against the homogeneous 'dense-vesicle' cyclic AMP phosphodiesterase from rat liver, identified a single 63 kDa species which was localized in the adipocyte endoplasmic reticulum fraction. The ability of adrenaline to stimulate adipocyte membrane cyclic AMP phosphodiesterase was shown to be mediated via beta-adrenoceptors and not alpha 1-adrenoceptors. Membrane cyclic AMP phosphodiesterase was stimulated by glucagon but not by vasopressin, A23187 or 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment of adipocytes with either chloroquine or dansyl cadaverine failed to affect the ability of insulin to stimulate cyclic AMP phosphodiesterase activity. Treatment of an isolated adipocyte endoplasmic reticulum membrane fraction with purified protein kinase A increased its cyclic AMP phosphodiesterase activity some 2-fold. When this fraction was treated with purified protein kinase A and [32P]ATP, label was incorporated into a 63 kDa protein which was specifically immunoprecipitated with the antiserum against the liver 'dense-vesicle' cyclic AMP phosphodiesterase.

Flow cytometry, a very useful technique for the characterization of intestinal membrane vesicles.

The plasma membrane of enterocytes comprises two structurally and functionally distinct domains. These are the apical brush border, containing digestive hydrolases and glycocalyx, and the basolateral domain, characterized by other specific markers. Using a fast and easy subcellular fractionation, we purified four membrane vesicle fractions from rabbit small intestinal mucosa: brush border, basolateral, rough endoplasmic reticulum and Golgi + smooth endoplasmic reticulum. Using flow cytometry, the fluorescence polarization of diphenylhexatriene was determined in brush border and in basolateral + Golgi + smooth endoplasmic reticulum membrane fractions in order to investigate changes in the membrane fluidity of both fractions and to compare the results obtained with those of spectroscopic techniques. Moreover, it was possible with flow cytometry to detect and quantify basolateral and brush border markers by using polyclonal and monoclonal antibodies. The advantages of flow cytometry in the detection of brush border membrane markers found in small amounts in the basolateral domain are discussed. Finally, flow cytometry holds great promise for the analysis and sorting of subcellular fractions.

The hydrolysis of the alkenyl group from enthanolamine-plasmalogen by oligodendroglial cell-enriched fractions

The rate of hydrolysis of the 1-0-alkenyl group of sn-1-alk-1?-enyl-2-acyl-glycerylphosphorylethanolamine (alkenyl, acyl-GPE; ethanolamine plasmalogen) by plasmalogenase is higher in oligodendroglial cell-enriched fractions from bovine brain compared with fractions enriched in neuronal perikarya and astroglia. The distribution of plasmalogenase activity in membrane fractions isolated from bovine oligodendroglia has been compared with that of 'marker' enzymes. The highest specific activity was in a fraction enriched in plasma membranes, whilst most activity was recovered in an endoplasmic reticulum membrane fraction. In bovine oligodendroglial cell homogenates, the enzyme had a neutral pH optimum, had no requirement for divalent cations and its activity towards 1-alkenyl-GPE (lysoplasmalogen) was half that with alkenyl, acyl-GPE. C(16) alkenyl groups were hydrolysed more rapidly than C(18) alkenyl groups. With (3)H-labelled alkenyl, acyl-GPE as substrate, radioactivity in released aldehydes appeared in fatty acids esterified in phospholipid while the oxidation of fatty aldehydes was blocked by the addition of NADH. An NAD-dependent aldehyde dehydrogenase was found to be present in oligodendroglia which exhibited highest activity towards C(14)?C(18) aldehydes (K(m), 2 ?M).

Suitability of L-[35S]methionine for studying the biosynthesis of the polypeptides of mouse liver endoplasmic reticulum membrane fractions in vivo.

The use of L-[35S]methionine (500-700 Ci/mmol (1 Ci = 37 GBq) for labelling the polypeptides of liver rough (R) and smooth (S)endoplasmic reticulum (ER) membrane fractions in vivo was studied. Adult mice were injected intraperitoneally with 400 muCi of the isotope and killed at various times (2'min to 24 h) thereafter. RER and SER fractions were prepared, stripped of ribosomes, and treated with Triton X-100 to remove intravesicular contents. Sufficient radioactivity was present in individual aliquots (75 microgram protein) of the ER membrane fractions to permit their analysis by fluorography after separation by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. By 3 min, although the majority of the labelled components were of intravesicular origin, some 12 membrane polypeptides were labelled in the RER fraction (including one corresponding in migration to cytochrome P-450); some 6 of these latter polypeptides were labelled to a lesser degree in the SER membrane fraction at this time. By 5 min, the patterns of radioactive polypeptides of the RER and SER fractions (including both membrane and intravesicular components) were identical. By 7 min, some 28 labelled membrane polypeptides were detectable in the total microsomal membrane. Analysis of the 24-h samples revealed that all the membrane polypeptides seen by staining with Coomassie blue were visualised by fluorography. Other studies revealed the applicability of the approach used for producing highly labelled cell sap and serum proteins. The overall results demonstrate the suitability of L-[35S]methionine administered in vivo for producing mouse liver ER membrane polypeptides of relatively high radioactivity and are consistent with a rapid conversion of RER to SER by ribosome detachment or membrane flow.

Electrophoretic studies on liver endoplasmic reticulum membrane polypeptides and on their phosphorylation in vivo and in vitro.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to examine the polypeptide patterns of rat liver rough and smooth endoplasmic reticulum (ER) membrane fractions stripped of ribosomes. Approximately 67 polypeptides were resolved from the rough ER membrane fraction. The polypeptide pattern of the smooth ER membrane fraction was similar to that of the rough ER membrane fraction, but exhibited substantially lower amounts of some seven polypeptides. Three of these polypeptides, of apparent molecular weights 63,000, 65,000, and 87,000, were of particular interest, as they could not be ascribed to contamination of stripped rough ER membrane fractions by residual ribosomal polypeptides. Conditions of treatment with low concentrations of trypsin were established that markedly diminished the capacity of the stripped rough ER membrane fraction to bind ribosomes in vitro and that also effected a partial detachment of ribosomes from nonstripped rough ER membranes; the results of electrophoretic analyses of rough ER membrane fractions treated in these manners are described. Comparison of the polypeptide patterns of guinea pig, mouse, and rabbit liver ER membrane fractions with rat liver ER membrane fractions revealed considerable variations in the distribution of the polypeptides of 63,000, 65,000, and 87,000 molecular weight among the ER membrane fractions of these species. The combined results of these studies indicate that the polypeptide of 87,000 molecular weight, although particularly sensitive to attack by trypsin, is not involved in the binding of ribosomes to the rough ER membrane fraction. Studies by others (cf. Kreibich, G., Grebenau, R., Mok, W., Pereyra, B., Rodriguez-Boulan, E., and Sabatini, D. D. (1977) Fed. Proc. 36, 656) have implicated the polypeptides of 63,000 and 65,000 molecular weight in this process. The patterns of phosphorylated polypeptides of rough and smooth ER membrane fractions of rat and mouse liver were also examined, using labeling in vivo with sodium [32p]phosphate or in vitro with [gamma-32P]ATP. Approximately 25 phosphorylated components were resolved by electrophoresis in the ER membrane fractions of both species. Evidence is presented that suggests that the great majority of these components are phosphopolypeptides. Differences were noted in the patterns of phosphorylation produced by in vivo and in vitro labeling; minor differences were also observed between the patterns of phosphorylation of the rough and smooth ER membrane fractions in either situation. The overall results afford an indirect approach toward evaluating the possible involvement of specific rough ER membrane polypeptides in ribosome-binding and reveal that liver ER membranes contain a substantially greater number of phosphorylated polypeptides thatn previously reported.

Phospholipid Synthesis and Exchange in Castor Bean Endosperm Homogenates

Crude organelle preparations from castor bean (Ricinus communis L.) endosperm rapidly incorporate CDP-((14)C)choline and CDP-((14)C)-ethanolamine into phosphatidylcholine and phosphatidylethanolamine, respectively. Separation of organelles by sucrose density gradient centrifugation following incubation with these substrates demonstrated that most of the (14)C phospholipids thus formed were present in the endoplasmic reticulum membranes, although label was also found in mitochondria, proplastids, and glyoxysomes. The phospholipid-synthesizing enzymes, cholinephosphotransferase and ethanolaminephosphotransferase, are exclusively confined to the endoplasmic reticulum membrane fraction, suggesting that the appearance of (14)C-phospholipid in other organelles was due to phospholipid exchange. Phospholipid synthesis was inhibited by the cytoplasmic supernatant fraction. The active inhibitor in this fraction was not identified, but the inhibition was not significantly relieved by either dialyzing or boiling the supernatant. Phosphatidylcholine synthesis showed an absolute requirement for Mg(2+); the Michaelis constant was 1 mm. Ca(2+) was a potent inhibitor of Mg(2+)-stimulated phospholipid synthesis and enhanced the decay of (14)C-phospholipids from pre-labeled membranes, particularly when the membranes were resuspended in the cytoplasmic supernatant.The data are consistent with the concept that the endoplasmic reticulum is a major site of membrane proliferation where structural lipids, and possibly proteins, are inserted into, and thus expand, a pre-existing membrane fraction. Other organelle and cellular membranes could therefore originate from the proliferating endoplasmic reticulum by a process of membrane flow and differentiation.

Norepinephrine-responsive beta-adrenergic receptors in rabbit lacrimal gland.

ARTICLESNorepinephrine-responsive beta-adrenergic receptors in rabbit lacrimal glandSY Botelho, AM Goldstein, and EV MartinezSY Botelho, AM Goldstein, and EV MartinezPublished Online:01 May 1973https://doi.org/10.1152/ajplegacy.1973.224.5.1119MoreSectionsPDF (862 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited Byβ-Adrenoreceptors as Therapeutic Targets for Ocular Tumors and Other Eye Diseases—Historical Aspects and Nowadays Understanding28 February 2023 | International Journal of Molecular Sciences, Vol. 24, No. 5A Lacrimal Gland is a Lacrimal Gland, But Rodent's and Rabbit's Are Not HumanThe Ocular Surface, Vol. 8, No. 3Hormonal influence on expression and functionality of α1-adrenoceptor in rat submandibular glandAutonomic Neuroscience, Vol. 103, No. 1-2Exocrine Gland Dysfunction in MC5-R-Deficient Mice: Evidence for Coordinated Regulation of Exocrine Gland Function by Melanocortin PeptidesCell, Vol. 91, No. 6Protein phosphorylation in Golgi, endosomal, and endoplasmic reticulum membrane fractions of lacrimal gland2 July 2009 | Current Eye Research, Vol. 15, No. 2Hormonal Support of Lacrimal Function, Primary Lacrimal Deficiency, Autoimmunity, and Peripheral Tolerance in the Lacrimal Gland8 July 2009 | Ocular Immunology and Inflammation, Vol. 4, No. 3Fluid phase endocytosis by isolated rabbit lacrimal gland acinar cellsExperimental Eye Research, Vol. 60, No. 5Effect of topically applied oxymetazoline on tear volume and tear flow in humans1 March 1991 | Graefe's Archive for Clinical and Experimental Ophthalmology, Vol. 229, No. 2Yohimbine and lacrimal secretion [letter]26 July 2012 | British Journal of Clinical Pharmacology, Vol. 30, No. 1The Protective Mechanism?-Adrenoceptors in the extraorbital lacrimal gland of the Syrian hamster characterization with [125I]-iodopindolol and evidence of sexual dimorphismJournal of Neural Transmission, Vol. 77, No. 2-3Lacrimal fluid and electrolyte secretion: A review2 July 2009 | Current Eye Research, Vol. 8, No. 6Signal transduction and control of lacrimal gland protein secretion: A review2 July 2009 | Current Eye Research, Vol. 8, No. 6Vasoactive intestinal peptide activates Ca2+ -dependent K+ channels through a cAMP pathway in mouse lacrimal cellsNeuron, Vol. 1, No. 3Eyelid secretions and the prevention and production of diseaseEye, Vol. 2, No. 2Distribution of adrenergic nerves in the lacrimal glands of guinea-pig and ratExperimental Eye Research, Vol. 40, No. 5Stimulation of lacrimal secretion by sympathetic nerve impulses in the rabbitLife Sciences, Vol. 34, No. 24Adrenergic control of lacrimal secretion in rabbitsLife Sciences, Vol. 34, No. 24Autonomic Nervous System: Adrenergic AgonistsAutonomic Nervous System: Adrenergic AntagonistsAdrenocorticotropic Hormone and alpha-Melanocyte-Stimulating Hormone Induce Secretion and Protein Phosphorylation in the Rat Lacrimal Gland by Activation of a cAMP-Dependent PathwayEuropean Journal of Biochemistry, Vol. 126, No. 3β-ReceptorenblockerSecretory effect of prostaglandins on the rabbit lacrimal glandProstaglandins and Medicine, Vol. 3, No. 3INHIBITION AND FACILITATION OF LACRIMAL FLOW BY β-ADRENERGIC DRUGS27 May 2009 | Acta Ophthalmologica, Vol. 57, No. 2RESULTS FROM A MULTICENTRE TRIAL OF METOPROLOL AND A STUDY OF HYPERTENSIVE PATIENTS WITH CHRONIC OBSTRUCTIVE LUNG DISEASE1 March 1978 | Medical Journal of Australia, Vol. 1, No. 5The Lacrimal GlandSide effects and contraindications of beta-receptor blocking agentsKlinische Wochenschrift, Vol. 55, No. 9 More from this issue > Volume 224Issue 5May 1973Pages 1119-1122 Copyright & PermissionsCopyright © 1973 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1973.224.5.1119PubMed4144765History Published online 1 May 1973 Published in print 1 May 1973 Metrics