Asymmetric reconstitution of homogeneous Escherichia coli sn-glycerol-3-phosphate acyltransferase into phospholipid vesicles

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane was purified in Triton X-100 (Green, P. R., Merrill, A. H., Jr., and Bell, R. M. (1981) J. Biol. Chem. 256, 11151-11159) and incorporated into mixed micelles containing Triton X-100, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and beta-octyl glucoside. Enzyme activity was quantitatively reconstituted from the mixed micelle into single-walled phospholipid vesicles by chromatography over Sephadex G-50. Activity coeluted with vesicles of 90-nm average diameter on columns of Sepharose CL-4B and Sephacryl S-1000. These vesicles contained less than 2 Triton X-100 and 5 beta-octyl glucoside molecules/100 phospholipid molecules. Calculations suggested that up to eight 91,260-dalton glycerol-P acyltransferase polypeptides were incorporated per 90-nm vesicle. The pH dependence and apparent Km values for glycerol-P and palmitoyl-CoA of the glycerol-P acyltransferase reconstituted into vesicles were similar to those observed upon reconstitution by mixing of the enzyme in Triton X-100 with a 20-fold molar excess of sonicated phosphatidylethanolamine:phosphatidylglycerol:cardiolipin, 6:1:1. The integrity of vesicles containing glycerol-P acyltransferase was established by trapping 5,5'-dithiobis-(2-nitrobenzoic acid). Chymotrypsin inactivated greater than 95% of the glycerol-P acyltransferase in intact vesicles and cleaved the 91,260-dalton polypeptide into several vesicle-bound and several released peptides, indicating that critical domains of the enzyme are accessible in intact vesicles. Trinitrobenzene sulfonate and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene caused greater than 90% loss of glycerol-P acyltransferase in vesicles. Disruption of vesicles with Triton X-100 did not reveal significant latent activity. These data strongly suggest that the glycerol-P acyltransferase was reconstituted asymmetrically into the vesicles with its active site facing outward.     

sn-glycerol-3-phosphate acyltransferase tubule formation is dependent upon heat shock proteins (htpR)

Overexpression of the Escherichia coli sn-glycerol-3-phosphate (glycerol-P) acyltransferase, an integral membrane protein, causes formation of ordered arrays of the enzyme in vitro. The formation of these tubular structures did not occur in an E. coli strain bearing a mutation in the htpR gene, the regulatory gene for the heat shock response. The htpR165 mutation was shown by genetic analysis to be the lesion responsible for blockage of tubule formation. Similar amounts of glycerol-P acyltransferase were produced in isogenic htpR+ and htpR165 strains, ruling out an effect of htpR165 on expression of glycerol-P acyltransferase. Further, phospholipid metabolism was not altered in either strain after induction of glycerol-P acyltransferase synthesis. Increased glycerol-P acyltransferase synthesis caused a partial induction of the heat shock response which was dependent upon a wild type htpR gene. The heat shock proteins induced were identified as the groEL and dnaK gene products on two-dimensional gels. These two proteins have been implicated in the assembly of bacteriophage coats. These heat shock proteins appear essential for tubule formation.     

Efficiency of reconstitution of the membrane-associated sn-glycerol 3-phosphate acyltransferase of Escherichia coli

Membrane-associated enzymes are often solubilized with detergents, purified, and then reconstituted with phospholipid cofactors to regain function. Insofar as most purification and reconstitution procedures are not quantitative, the final reconstituted preparations could reflect a population of molecules ranging from fully functional to completely inactive. Quantitative studies on the efficiency of reconstitution of the Triton X-100-solubilized sn-glycerol 3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane were undertaken at each step of purification. Physical recovery of the 83,000 Mr polypeptide was quantitated in polyacrylamide gels using membranes from cells labeled with [3H]leucine. The 83,000 Mr polypeptide in such gels was demonstrated to consist exclusively of the glycerol-P acyltransferase peptide by V8 peptide mapping. Comparison between physical recovery of 83,000 Mr polypeptide and reconstituted activity allowed the efficiency of reconstitution to be determined. Unexpectedly, disproportionalities occurred during the purification. However, the final purification of reconstituted enzyme activity matched that of the 83,000 Mr polypeptide. This method also allowed measurement of the specific activities of the glycerol-P acyltransferase in membranes from a wild type E. coli strain and from plasmid-containing strains which express the plsB gene product to different extents. The physical amounts of the 83,000 Mr polypeptide and glycerol-P acyltransferase activity measured in membranes were not strictly proportional. In strains where the amount of 83,000 Mr polypeptide was enhanced, a larger proportion of latent activity was observed following solubilization and reconstitution. The results establish the suitability of the reconstituted preparations of glycerol-P acyltransferase for detailed kinetic analysis and permit inferences pertaining to regulation.     

Isolation and characterization of the tubular organelles induced by fumarate reductase overproduction in Escherichia coli

Strains of Escherichia coli amplifying the intrinsic membrane enzyme fumarate reductase accommodate the overproduced enzyme by increasing the amount of membrane material, in the form of intracellular tubular structures. These tubules have been observed in strains harbouring multicopy frd plasmids and in ampicillin hyper-resistant strains. A procedure has been developed for isolation of tubules nearly free of cytoplasmic membrane. Using protein A-gold labelling and optical diffraction of electron micrographs, a model for tubule structure is proposed. The tubules have a lower lipid/protein ratio than the cytoplasmic membrane, with the enzyme accounting for greater than 90% of the protein in the tubules. Both cytoplasmic membranes and tubules from amplified strains are enriched in cardiolipin and have a more fluid fatty acid composition than wild-type strains. Mutants defective in cardiolipin synthesis produce tubules in response to excess fumarate reductase, but these tubules have an altered appearance, indicating that lipid-protein interactions may be important for tubule assembly.     

Inhibition of a Golgi complex lysophospholipid acyltransferase induces membrane tubule formation and retrograde trafficking

Recent studies have suggested that formation of Golgi membrane tubules involves the generation of membrane-associated lysophospholipids by a cytoplasmic Ca2+-independent phospholipase A2 (PLA2). Herein, we provide additional support for this idea by showing that inhibition of lysophospholipid reacylation by a novel Golgi-associated lysophosphatidylcholine acyltransferase (LPAT) induces the rapid tubulation of Golgi membranes, leading in their retrograde movement to the endoplasmic reticulum. Inhibition of the Golgi LPAT was achieved by 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976), a previously characterized antagonist of acyl-CoA cholesterol acyltransferase. The effect of CI-976 was similar to that of brefeldin A, except that the coatomer subunit beta-COP remained on Golgi-derived membrane tubules. CI-976 also enhanced the cytosol-dependent formation of tubules from Golgi complexes in vitro and increased the levels of lysophosphatidylcholine in Golgi membranes. Moreover, preincubation of cells with PLA2 antagonists inhibited the ability of CI-976 to induce tubules. These results suggest that Golgi membrane tubule formation can result from increasing the content of lysophospholipids in membranes, either by stimulation of a PLA2 or by inhibition of an LPAT. These two opposing enzyme activities may help to coordinately regulate Golgi membrane shape and tubule formation.     

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Properties of wild type and Km defective sn-glycerol-3-phosphate acyltransferase activities.

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase, the first enzyme of membrane phospholipid synthesis in Escherichia coli, was investigated in a wild type and a mutant strain defective in this activity. The mutant strain, selected as a glycerol-P auxotroph, was previously shown to contain a glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of its parent or revertants. The membranous mutant glycerol-P acyltransferase but did not appear to be thermolabile in vivo. Revertants no longer requiring glycerol-P for growth, showed glycerol-P acyltransferase activity with thermolability properties similar to the wild type. The second phospholipid biosynthetic enzyme, 1-acylglycerol-P acyltransferase, was not thermolabile in membranes containing a thermolabile glycerol-P acyltransferase activity. The pH optimum for the mutant acyltransferase was over 1 pH unit higher than that of the parental activity. Further, the mutant and wild type glycerol-P acyltransferase differed in their response to magnesium chloride and potassium chloride. The palmitoyl-CoA dependence of the wild type and mutant glycerol-P acyltransferase activities were different. The mutant glycerol-P acyltransferase activity was inhibited greater than 90% by Triton X-100 under conditions where the wild type activity was not affected. These experiments provide novel information about the wild type glycerol-P acyltransferase activity of E. coli and provide six additional lines of evidence for the mutant character of the glycerol-P acyltransferase in the mutant strains.     

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Phenotypic suppression of sn-glycerol-3-phosphate acyltransferase Km mutants by loss of feedback inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Revertants of Escherichia coli mutants defective in the first enzyme of membrane phospholipid synthesis, sn-glycerol-3-phosphate (glycerol-P) acyltransferase, were investigated. These glycerol-P acyltransferase mutants, selected as glycerol-P auxotrophs, contained membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than the parental activity. The glycerol-P acyltransferase activity was also more thermolabile in vitro than the parental activity. Most revertants no longer requiring glycerol-P for growth regained glycerol-P acyltransferase activity of normal thermolability and apparent Km for glycerol-P. However, two novel revertants were isolated which retained an abnormal glycerol-P acyltransferase activity. The glycerol-P dehydrogenase activities of these novel revertants were about 20-fold less sensitive to feedback inhibition by glycerol-P. The feedback-resistant glycerol-P dehydrogenase co-transduced with gpsA, the structural gene for the glycerol-P dehydrogenase. Further transduction experiments demonstrated that the feedback resistant glycerol-P dehydrogenase phenotypically suppressed the glycerol-P acyltransferase Km lesion. The existence of the class of glycerol-P auxotrophs which owe their phenotype to the glycerol-P acyltransferase Km lesion therefore depends on the feedback regulation of glycerol-P synthesis in E. coli.     

Triacylglycerol synthesis in isolated fat cells. Evidence that the sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities are dual catalytic functions of a single microsomal enzyme.

The acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (glycerol-P acyltransferase) and acyl-CoA:dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) (DHAP acyltransferase) activities were investigated in vitro in order to evaluate the quantitative contribution of the glycerol-P and DHAP pathways for the synthesis of triacylglycerols in isolated fat cells and to test the hypothesis that these two activities may be dual catalytic functions of a single enzyme. More than 85% of both acyltransferase activities was associated with the microsomal subcellular fraction. The microsomal glycerol-P acyltransferase activity showed an apparent Km of 8 muM for glycerol-P with a Vmax of 15.6 nmol/min/mg, while the DHAP acyltransferase activity showed an apparent Km of 40 muM for DHAP with a Vmax of 9.7 nmol/min/mg. Glycerol-P was a competitive inhibitor (Ki = 7.2 muM) of the DHAP acyltransferase, and DHAP was a competitive inhibitor (Ki = 92 muM) of the glycerol-P acyltransferase. The two acyltransferase activities showed virtual identity in their pH dependence, acyl-CoA chain length dependence, thermolability, and inactivation by N-ethylmaleimide. Trypsin, detergents, collagenase, phospholipases, and various salts and organic solvents also had similar effects on both activities. Taken as a whole, the data strongly suggest that the microsomal glycerol-P and DHAP acyltransferase activities actually represent dual functions of a single enzyme. Calculations based on the above kinetic constants and previously reported glycerol-P and DHAP pools in adipocytes suggest that the in vivo ratio of glycerol-P to DHAP acylation should be greater than 24:1.     

Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities.

Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAP acyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activity and that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a single membranous enzyme, (ii) estimate the relative contributions of the glycerol-P and DHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitability of yeast for future genetic investigations of the eucaryotic glycerol-P and DHAP acyltransferase activities. The membranous DHAP acyltransferase activity showed an apparent Km of 0.79 mM for DHAP, with a Vmax of 5.3 nmol/min per mg, whereas the glycerol-P acyltransferase activity showed an apparent Km of 0.05 mM for glycerol-P, with a Vmax of 3.4 nmol/min per mg. Glycerol-P was a competitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki, 0.91 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities exhibited marked similarities in their pH dependence, acyl-coenzyme A chain length preference and substrate concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Thus, the data strongly suggest that yeast glycerol-P and DHAP acyltransferase activities represent dual catalytic functions of a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxidoreductase activity could be detected in yeast membranes, the DHAP pathway for glycerolipid synthesis may not operate in yeast.     

Hepatic sn-glycerol-3-phosphate acyltransferases: effect of monoacylglycerol analogs on mitochondrial and microsomal activities

The regulation of cellular diacylglycerol levels may have important consequences for protein kinase C activity. Because monoacylglycerols were said to inhibit the committed step of glycerolipid synthesis, the sn-glycerol-3-P acyltransferase (glycerol-P acyltransferase), we determined (1) whether both the mitochondrial and the microsomal glycerol-P acyltransferase isoenzymes were inhibited by 1- and 2-mono-18:1-glycerols, and their ether and amide analogs and (2) what the mechanism of inhibition was. 1- and 2-mono-18:1-glycerols, their ether and amide analogs, and 1-mono-18:1-glycerol 3-phosphate were all competitive inhibitors of the microsomal glycerol-P acyltransferase activity. The relative Ki values suggested that inhibition was strongest with the radyl group at the sn-1 position and that an oxygen bond is important at the sn-1 position. Although the monoacyl- and monoalkylglycerols were also competitive inhibitors of the mitochondrial glycerol-P acyltransferase, neither of the amide analogs was an inhibitor, suggesting that an oxygen bond is essential at both the sn-1 and sn-2 positions. Because monoradylglycerols inhibit several enzyme activities that contribute to the biosynthesis or the metabolism of diacylglycerol, these inhibitors may function within cells in part to regulate cellular diacylglycerol levels.     

Isolation and characterization of membranes from a hydrocarbon-oxidizing Acinetobacter sp.

Membranes were isolated and purified from nutrient broth-yeast extract- and hexadecane-grown cells of Acinetobacter sp. strain HO1-N. Two membrane fractions were isolated from nutrient broth-yeast extract-grown cells, the cytoplasmic membrane and the outer membrane. In addition to these two membrane fractions, a unique membrane fraction was isolated from hexadecane-grown cells (band 1) and characterized as a lipid-rich, low-density membrane containing high concentrations of hexadecane. The outer membrane preparations of Acinetobacter, obtained from nutrient broth-yeast extract- and hexadecane-grown cells, exhibited a low ratio of lipid phosphorus to protein and contained phospholipase activity and 2-keto-3-deoxyoctulosonic acid. Phosphatidic acid cytidyltransferase, adenosine triphosphatase, and reduced nicotinamide adenine dinucleotide oxidase were recovered almost exclusively in the cytoplasmic membrane fractions. The cytoplasmic membrane fractions contained 20 to 25 polypeptide species on sodium dodecyl sulfate-polyacrylamide gels, and the outer membrane fractions contained 15 to 20 polypeptide species. A major polypeptide species with an apparent molecular weight of approximately 42,000 to 44,000 was found for all outer membrane fractions. The buoyant densities of the cytoplasmic membrane fractions and the outer membrane fractions were closely similar, necessitating their separation by differential centrifugation. Band 1 of hexadecane-grown cells had a ratio of lipid phosphorus to protein that was almost twice that of cytoplasmic membrane and a correspondingly low buoyant density (1.086 g/cm3). Enzyme activities associated with band 1 were identical to those associated with the cytoplasmic membrane. The electrophoretic banding pattern of band 1 was essentially identical to the banding pattern of the cytoplasmic membrane. The phospholipid and neutral lipid compositions of the isolated membrane fractions were determined as qualitatively similar, with significant quantitative differences. The ultrastructure characteristics of the respective membrane fractions were examined by the negative-stain technique.     

Identification of low molecular weight GTP-binding proteins and their sites of interaction in subcellular fractions from skeletal muscle

The presence of low molecular weight GTP-binding proteins was investigated in subcellular fractions from skeletal muscle. Skeletal muscle homogenate, transverse tubules, triads, sarcoplasmic reticulum membranes, and cytosol fractions were separated in sodium dodecyl sulfate-gel electrophoresis and blotted onto nitrocellulose. The presence of GTP-binding proteins was explored by incubation of these blots with [alpha-32P] GTP. GTP labeled two polypeptides of Mr = 23,000 and 29,000 in all the fractions examined. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 29-kDa polypeptide, although both were enriched in transverse tubule fractions. A GTP-binding polypeptide of 40 kDa was also enriched in transverse tubule preparations and identified as Gi alpha by immunostaining with anti-Gi alpha. Using a blot overlay approach and [alpha-32P]GTP-labeled cytosolic components, several polypeptides were identified that interact with the 23- and 29-kDa GTP-binding proteins. Among these components were polypeptides of Mr = 60,000, 47,000, 44,000, 42,000, and 38,000, which were mainly of cytosolic origin but also associated with triads and transverse tubule membranes. The 47-, 44-, 42-, and 38-kDa polypeptides were found to be structurally related to the glycolytic enzymes enolase, 3-phosphoglyceric phosphokinase, aldolase, and glycoeraldehyde-3-phosphate dehydrogenase, respectively. The purified glycolytic enzymes specifically bound the 23- and 29-kDa GTP-binding proteins under both denaturing and nondenaturing conditions. The association of the GTP-binding proteins with these polypeptides was resistant to detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), Triton X-100, and Tween. A 23-kDa GTP-binding protein purified from chromaffin cells bound to a 157-kDa polypeptide in triads and chromaffin cell membranes. The 157-kDa polypeptide was a minor component in these membranes and not related to the subunits of the dihydropyridine receptor. In view of the proposed function of low molecular weight GTP-binding proteins in processes such as membrane communication and secretion coupling, the association of these proteins with transverse tubules and triads in skeletal muscle is discussed in terms of a role in signal transmission.     

Regulation of the Golgi complex by phospholipid remodeling enzymes

The mammalian Golgi complex is a highly dynamic organelle consisting of stacks of flattened cisternae with associated coated vesicles and membrane tubules that contribute to cargo import and export, intra-cisternal trafficking, and overall Golgi architecture. At the morphological level, all of these structures are continuously remodeled to carry out these trafficking functions. Recent advances have shown that continual phospholipid remodeling by phospholipase A (PLA) and lysophospholipid acyltransferase (LPAT) enzymes, which deacylate and reacylate Golgi phospholipids, respectively, contributes to this morphological remodeling. Here we review the identification and characterization of four cytoplasmic PLA enzymes and one integral membrane LPAT that participate in the dynamic functional organization of the Golgi complex, and how some of these enzymes are integrated to determine the relative abundance of COPI vesicle and membrane tubule formation. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Selective changes in enzymes of the sn-glycerol 3-phosphate and dihydroxyacetone-phosphate pathways of triacylglycerol biosynthesis during differentiation of 3T3-L1 preadipocytes

Enzyme activities of the sn-glycerol 3-phosphate (glycerol-P) and of the dihydroxyacetone-phosphte (DHAP) pathway of glycerolipid biosynthesis were investigated during the differentiation of 3T3-L1 preadipocytes into adipocytes. Total particulate glycerol-P and DHAP acyltransferase activities increased 70- and 30-fold, respectively, during differentiation induced with methylisobutylxanthine and dexamethasone. The N-ethylmaleimide-sensitive (microsomal) glycerol-P and DHAP acyltransferase activities were virtually undetectable in nondifferentiated cells, and increased in parallel over 70-fold during differentiation. These and several kinetic observations are consistent with the induction of a single microsomal enzyme having dual activity. During differentiaion, the N-ethylmaleimide-resistant DHAP acyltransferase activity increased 10-fold, suggesting the presence of at least two DHAP acyltransferase isoenzymes. Qualitatively similar changes in microsomal glycerol-P and DHAP acyltransferase activities were observed when cell differentiation was induced with insulin or with insulin plus dexamethasone and methylisobutylxanthine. Acyl-DHAP oxidoreductase (EC 1.1.1.101) specific activity increased only 3- to 5-fold during adipocyte differentiation. Alkyl-DHAP synthase activity was not detected. These data demonstrate that selective changes in enzyme activities of the gycerol-P pathways of glycerolipid synthesis occur during the differentiation of 3T3-L1 preadipocytes.     

Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme.     

Structural characterization of ordered arrays of sn-glycerol-3-phosphate acyltransferase from Escherichia coli.

Overproduction of the sn-glycerol-3-phosphate acyltransferase in Escherichia coli leads to incorporation of this integral membrane protein into ordered tubular arrays within the cell. Freeze-fracture-etch shadowing was performed on suspensions of partially purified tubules and whole bacteria. This procedure revealed the presence of ridges and grooves defining a set of long-pitch left-handed helical ridges. The long-pitch helices represented chains of acyltransferase dimers. Tubules observed within the cell were often closely packed, with an apparent alignment of grooves and ridges in adjacent tubules. Fracture planes passing through the tubules indicated the presence of a bilayer structure, with some portion of the enzyme being associated with the membrane. The major portion of the enzyme extended from the hydrophilic surface, forming a large globular structure that, in favorable views, displayed a central cavity facing the cytoplasm. Computer analysis of shadowed tubules revealed that the left-handed helices were six stranded, with a pitch of 1,050 A (105.0 nm) and a spacing of 75 A (7.5 nm) between acyltransferase dimers along the chains. Analysis of the predicted secondary structure failed to reveal obvious transmembrane segments, suggesting that very little of the protein was inserted into the bilayer.     

Identification and purification of a transverse tubule coupling protein which binds to the ryanodine receptor of terminal cisternae at the triad junction in skeletal muscle

In fast twitch skeletal muscle, the signal for excitation-contraction coupling is transferred from transverse tubule across the triad junction; calcium is thereby released from the terminal cisternae of sarcoplasmic reticulum triggering muscle contraction. Recently, the feet structures of terminal cisternae, which bridge the gap at the triad junction, have been identified as the ryanodine receptor and in turn with the calcium release channels of sarcoplasmic reticulum. The latter consists of an oligomer of a single high molecular weight polypeptide (Mr 360,000). This study attempts to identify the component in the transverse tubule which ligands with the foot structure to form the triad junction. The purified ryanodine receptor, derivatized with sulfosuccinimidyl-2-(p-azidosalicylimido)-1,3'-dithiopropionate (SASD), a thiol-cleavable, 125I-iodinatable, and photoactive probe, was shown to selectively cross-link to a protein with Mr of 71,000 in isolated transverse tubules. This coupling protein was purified from transverse tubule by solubilization with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and then purified by sequential column chromatography. In the absence of sulfhydryl agents, the purified polypeptide has an Mr of 61,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A complementary approach using SASD was employed to confirm association of the coupling protein with the ryanodine receptor of terminal cisternae. We conclude that the transverse tubule coupling protein together with the ryanodine receptor (foot structure) is involved in the liganding between transverse tubule and terminal cisternae of sacroplasmic reticulum.     

Mammalian dynamin-like protein DLP1 tubulates membranes

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) but not GTP. EM of the GTPgammaS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPgammaS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.     

A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells

Abstract. Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.     

Hepatic monoacylglycerol acyltransferase: ontogeny and characterization of an activity associated with the chick embryo

Hepatic monoacylglycerol acyltransferase is expressed during the perinatal period in rats and guinea pigs and appears to be related temporally to the availability of fatty acids and to the development of hepatic steatosis. In order to determine when monoacylglycerol acyltransferase activity is expressed in an avian species, its ontogeny was investigated in chick liver total particulate preparations. In livers from 11- to 21-day-old chick embryos, monoacylglycerol acyltransferase specific activity was 34.5 +/- 8.1 nmol/min per mg of total particulate protein. The specific activity decreased 93% to 2.6 +/- 1.3 nmol/min per mg by the 6th day after hatching. The specific activities of fatty acid CoA ligase, diacylglycerol acyltransferase, and microsomal and mitochondrial glycerol-P acyltransferases changed comparatively little during this time period. In the embryos, the monoacylglycerol acyltransferase activity per liver rose 28-fold between the 11th and 21st day, corresponding exactly to the increase in liver total particulate protein during this time. Monoacylglycerol acyltransferase activity in other tissues was 25- to 115-fold lower than observed in liver. Optimal activity was measured using 25 microM palmitoyl-CoA and 50 microM sn-2-monooleoylglycerol. The activity with the 1- and 2-monooleoylglycerol ethers and 1-monooleoylglycerol was very low. In contrast to microsomes from rat liver, about 70% of the product with the 1- and 2-monooleoylglycerol ethers was triradylglycerol, suggesting that the diacylglycerol acyltransferase from chick liver can acylate acyl, alkylglycerols. The activity with sn-2-monooleoylglycerol amide was 12.5% of that observed with the corresponding 2-monooleoylglycerol suggesting that the ester bond is important; the 1-monooleoylglycerol amide was not a substrate.(ABSTRACT TRUNCATED AT 250 WORDS)