Localization and quantitation of proteins characteristic of the complexed membrane of Bacillus subtilis

We prepared antibodies to four proteins (molecular weights, 68,000, 64,000, 45,000, and 31,000) that are characteristic of the complexed (ribosome-bearing) fraction of the membrane of Bacillus subtilis and found that these proteins are immunologically distinct. Quantitation by immunoprecipitation confirmed that the ribosome-free membrane fraction contains much lower concentrations of these four proteins than the complexed-membrane fraction. The 64-kilodalton protein appeared to be attached more loosely than the other proteins, since it was more readily extracted from the membrane. In addition, this protein was also present in the cytosol in an even greater amount than in the membrane. The 68-, 64-, and 31-kilodalton proteins are present in cells in stoichiometrically equivalent amounts.     

ELECTRON MICROSCOPE EXAMINATION OF SUBCELLULAR FRACTIONS : III. Quantitative Analysis of the Microsomal Fraction Isolated from Rat Liver

Rat liver microsomes and microsomal subfractions isolated by density equilibration were submitted to a quantitative morphological and biochemical analysis. The total area of the endoplasmic reticulum was estimated at 7.3 m² per g of liver. The microsome fraction contained 2.8 mg of phospholipids and 6.7 mg of proteins per m² of membrane area. After correction for ribosomal and intracisternal proteins, the latter value was lowered to 4.7 mg of membrane protein per m². More than half of the microsomal vesicles carried ribosomes. After density equilibration of the microsomes, the distribution pattern of ribosomes followed closely that of RNA. The ribosome load of the microsomal vesicles increased steadily along the density gradient, indicating the existence of a continuous spectrum of microsomal entities ranging from entirely ribosome-free vesicles to vesicles heavily coated with ribosomes.     

Uptake of tetracycline by membrane preparations from Escherichia coli

1. A membrane fraction from Escherichia coli has been prepared essentially free from ribosomes by treatment of the membranes with Triton X-100 at 0 degrees C followed by differential centrifugation. 2. The ribosome-free membrane vesicles absorbed tetracycline by a reversible temperature-dependent process with an apparent K(m) of 0.029mm at pH7.5 and 37 degrees C. 3. The absorption process was negligible below 25 degrees C and had an optimum at 40 degrees C; a pH optimum at 7.5 was observed. 4. The absorption of tetracycline was strongly inhibited by EDTA and ATP; ADP inhibited less strongly and AMP had no effect. 5. There was no significant difference in the rates or extent of uptake of tetracycline by membranes prepared from tetracycline-sensitive and tetracycline-resistant, R-factor-bearing E. coli.     

Specific inhibition of phosphatidate cytidylyltransferase from Bacillus subtilis membranes by cytidine monophosphate

In Bacillus subtilis, the phosphatidate cytidylyltransferase was localized exclusively in the membrane fraction prepared by sucrose density gradient fractionation. A single enzyme could synthesize the two liponucleotides: CDPdiacylglycerol and dCDPdiacylglycerol. Kinetic experiments and isotopic exchange reactions suggested a ping-pong mechanism. Among the nucleosides monophosphate, CMP specifically reduced the synthesis of both liponucleotides. This inhibition was non-competitive and might be involved in regulation of phospholipid synthesis.     

Identification of GABAA/Benzodiazepine Receptors on Clathrin-Coated Vesicles from Rat Brain

Abstract: To investigate the subcellular compartments that are involved in the endocytosis and intracellular trafficking of GABA_A/benzodiazepine receptors, we have studied the distribution and properties of clonazepam-displaceable binding of [³H]flunitrazepam to membrane fractions from rat brain. The microsomal fraction was subjected to density centrifugation and gel filtration to isolate clathrin-coated vesicles. Homogeneity of the coated-vesicle fraction was demonstrated by using electron microscopy and by analysis of clathrin subunits and clathrin light-chain kinase. Vesicles exhibiting specific binding of [³H]flunitrazepam eluted from the sieving gel as a separate peak, which was coincident with that for coated vesicles. Scatchard analysis of equilibrium binding of [³H]flunitrazepam to coated vesicles yielded a K_D value of 21 ± 4.7 nM and a B_max value of 184 ± 28 fmol/mg. The K_D value for coated vesicles was 12-19-fold that found with microsomal or crude synaptic membranes. This low-affinity benzodiazepine receptor was not identified on any other subcellular fraction and thus appears to be a novel characteristic of coated vesicles. The B_maxvalue for coated vesicles, expressed per milligram of protein, corresponded to 16 and 115% of that found for crude synaptic and microsomal membrane fractions, respectively. Because the trafficking of neurotransmitter receptors via clathrin-coated vesicles is most likely to occur through endocytosis, the data suggest that an endocytotic pathway may be involved in the removal of GABA_A/benzodiazepine receptors from the neuronal surfaces of the rat brain. This mechanism could play a role in receptor sequestration and down-regulation that is produced by exposure to GABA and benzodiazepine agonists.     

Subcellular compartmentation of albumin and globin made in oocytes under the direction of injected messenger RNA

The Xenopus oocyte can be used to study the nature and specificity of the translational and post-translational systems of a normal living cell. We describe experiments combining messenger RNA microinjection and subcellular fractionation. Total Xenopus liver RNA directs the synthesis of albumin and vitellogenin contained within membrane vesicles; similarly, guinea pig mammary gland mRNA codes for membrane-bound protease-resistant milk proteins. In contrast, iodinated albumin protein injected into oocytes remains in the supernatant fraction, as does globin made on mRNA. The information encoded in the albumin messenger is therefore sufficient to specify synthesis of a membrane-bound product; moreoever, this highly efficient coupled translation-processing system may be of use in the study of the transfer of newly made proteins across membranes. A significant proportion (up to 20%) of newly made oocyte proteins enter a light membrane fraction, and many remain there. We speculate that these vesicles represent part of an important storage system.     

Differential distribution of voltage-dependent calcium channels and guanylate cyclase in the excitable ciliary membrane from Paramecium tetraurelia

A novel method for isolation of cilia and ciliary membrane vesicles from Paramecium tetraurelia has been developed. Using a continuous Percoll gradient of low osmolarity after fragmentation of purified cilia by French Press treatment two membrane fractions with different buoyant densities were obtained. These fractions were further purified by conventional discontinuous sucrose density gradients and characterized biochemically and by electron microscopy. Guanylate cyclase, a membrane bound enzyme, was found almost exclusively in membrane vesicles of high buoyant density while the voltage-sensitive calcium-channel of the ciliary membrane was predominantly localized in low density vesicles. Examination of both fractions by SDS polyacrylamide gel electrophoresis revealed only minor differences in protein pattern in the 34 and 64 kilodaltons range. Morphologically both membrane vesicle fractions had a diameter of about 300 nm, however, the high density vesicle fraction contained a considerably larger amount of multilamellar structures with a multishell, onion-like appearance. Freeze-fracture analysis failed to detect differences in intramembrane particle content between low and high density vesicles. The possible biological relevance of the spatial separation of the calcium-sensor enzyme guanylate cyclase and the voltage-sensitive calcium-channels in the ciliary membrane is discussed in terms of a diffusion controlled mechanism for graded signal transmission.     

Calcium sequestering activities of reticulum vesicles from Xenopus laevis oocytes

Membrane vesicles isolated from Xenopus laevis full-grown stage VI and mature oocytes accumulate 45Ca in the presence of ATP and oxalate. The Ca2+-pumping activity measured in vitro does not appear to be modified during meiotic maturation; it is not affected by the complex Ca2+-calmodulin. Preliminary experiments have shown that the addition of Na+ (30 mM) rapidly discharges accumulated 45Ca into oocyte vesicles indicating that a Na+/Ca2+ exchange system occurs in this membrane fraction. During progesterone-induced maturation, the different intracellular membranes undergo morphological changes. We suggest that intracellular movement of membrane vesicles could be involved in the local regulation of Ca2+ levels.     

Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport.

Syntaxins are thought to be membrane receptors that bind proteins of the synaptobrevin/vesicle-associated membrane protein (VAMP) family found on transport vesicles. Recently, we detected synaptobrevin II and cellubrevin on immunopurified vesicles containing the glucose transporter 4 (GLUT4) in insulin-responsive cells. In an effort to identify the plasma membrane receptors for these vesicles, we now examine the expression of syntaxins in the 3T3-L1 adipocyte cell line. Neither syntaxin 1A nor 1B was found, in keeping with the neuronal restriction of these isoforms. In contrast, syntaxins 2 and 4 were readily detectable. By subcellular fractionation and estimation of protein yields, 67% of syntaxin 4 was localized to the plasma membrane, 24% to the low-density microsomes, and 9% to the high-density microsomes. Interestingly, acute insulin treatment decreased the content of syntaxin 4 in low-density microsomes and caused a corresponding gain in the plasma membrane fraction, reminiscent of the recruitment of GLUT4 glucose transporters. In contrast, there was no change in the distribution of syntaxin 2, which was mostly associated in the plasma membrane. A fraction of the intracellular syntaxin 4 was recovered with immunopurified GLUT4-containing vesicles. Moreover, anti-syntaxin 4 antibodies introduced in permeabilized 3T3-L1 adipocytes significantly reduced the insulin-dependent stimulation of glucose transport, in contrast to the introduction of irrelevant immunoglobulin G, which was without consequence. We propose that either the plasma membrane and/or the vesicular syntaxin 4 are involved in docking and/or fusion of GLUT4 vesicles at the cell surface of 3T3-L1 adipocytes.     

Proteomics of neuroendocrine secretory vesicles reveal distinct functional systems for biosynthesis and exocytosis of peptide hormones and neurotransmitters

Regulated secretory vesicles produce, store, and secrete active peptide hormones and neurotransmitters that function in cell-cell communication. To gain knowledge of the protein systems involved in such secretory vesicle functions, we analyzed proteins in the soluble and membrane fractions of dense core secretory vesicles purified from neuroendocrine chromaffin cells. Soluble and membrane fractions of these vesicles were subjected to SDS-PAGE separation, and proteins from systematically sectioned gel lanes were identified by microcapillary LC-MS/MS (microLC-MS/MS) of tryptic peptides. The identified proteins revealed functional categories of prohormones, proteases, catecholamine neurotransmitter metabolism, protein folding, redox regulation, ATPases, calcium regulation, signaling components, exocytotic mechanisms, and related functions. Several novel secretory vesicle components involved in proteolysis were identified consisting of cathepsin B, cathepsin D, cystatin C, ubiquitin, and TIMP, as well carboxypeptidase E/H and proprotein convertases that are known to participate in prohormone processing. Significantly, the membrane fraction exclusively contained an extensive number of GTP nucleotide-binding proteins related to Rab, Rho, and Ras signaling molecules, together with SNARE-related proteins and annexins that are involved in trafficking and exocytosis of secretory vesicle components. Membranes also preferentially contained ATPases that regulate proton translocation. These results implicate membrane-specific functions for signaling and exocytosis that allow these secretory vesicles to produce, store, and secrete active peptide hormones and neurotransmitters released from adrenal medulla for the control of physiological functions in health and disease. In summary, this proteomic study illustrates secretory vesicle protein systems utilized for the production and secretion of regulatory factors that control neuroendocrine functions.     

Priming in exocytosis: attaining fusion-competence after vesicle docking

Membrane contact established by tethering or docking mechanisms is not a sufficient condition for membrane fusion. In neural and neuroendocrine cells, only a small fraction of secretory vesicles docked at the plasma membrane are fusion-competent and undergo rapid ATP-independent fusion in response to Ca(2+) elevations. Additional biochemical events termed 'priming' are essential to render vesicles competent for Ca(2+)-triggered fusion. The priming of vesicles is ATP-dependent and a number of ATP-dependent priming reactions have been characterized in permeable neuroendocrine cells. These involve NSF-mediated priming of SNARE protein complexes, the ATP-dependent synthesis of phosphoinositides, and protein kinase-mediated protein phosphorylation. In addition, munc13 is an important protein involved in priming synaptic vesicles. An emphasis in this review is on recent work indicating that priming events identified in the pathways of regulated exocytosis share many features with pre-fusion processes characterized in constitutive fusion pathways.     

Single Point Mutations Result in the Miss-Sorting of Glut4 to a Novel Membrane Compartment Associated with Stress Granule Proteins

Insulin increases cellular glucose uptake and metabolism in the postprandial state by acutely stimulating the translocation of the Glut4 glucose transporter from intracellular membrane compartments to the cell surface in muscle and fat cells. The intracellular targeting of Glut4 is dictated by specific structural motifs within cytoplasmic domains of the transporter. We demonstrate that two leucine residues at the extreme C-terminus of Glut4 are critical components of a motif (IRM, insulin responsive motif) involved in the sorting of the transporter to insulin responsive vesicles in 3T3L1 adipocytes. Light microscopy, immunogold electron microscopy, subcellular fractionation, and sedimentation analysis indicate that mutations in the IRM cause the aberrant targeting of Glut4 to large dispersed membrane vesicles that are not insulin responsive. Proteomic characterization of rapidly and slowly sedimenting membrane vesicles (RSVs and SSVs) that were highly enriched by immunoadsorption for either wild-type Glut4 or an IRM mutant revealed that the major vesicle fraction containing the mutant transporter (IRM-RSVs) possessed a relatively small and highly distinct protein population that was enriched for proteins associated with stress granules. We suggest that the IRM is critical for an early step in the sorting of Glut4 to insulin-responsive subcellular membrane compartments and that IRM mutants are miss-targeted to relatively large, amorphous membrane vesicles that may be involved in a degradation pathway for miss-targeted or miss-folded proteins or represent a transitional membrane compartment that Glut4 traverses en route to insulin responsive storage compartments.     

RNA replication of mouse hepatitis virus takes place at double-membrane vesicles

The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5'-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.     

Preparation of right-side-out plasma membrane vesicles from Penicillium cyclopium: a critical assessment of markers.

A plasma membrane fraction was obtained by the combined use of differential centrifugation and aqueous polymer two-phase partitioning techniques. Vanadate-inhibited ATPase and glucan synthase activities were highly enriched in this fraction, although the presence of ATPase activity which was not inhibited by vanadate, nitrate, molybdate, anyimycin A or azide was also detected. Other intracellular membrane marker activities were present at very low or undetectable levels. A further separation step using Percoll density gradient centrifugation resulted in the separation of a fraction which exclusively contained vanadate-inhibited ATPase activity, and was enriched with silicotungstic-acid-staining membrane material. Latency tests performed on the plasma membrane markers showed that the membrane vesicles were in the right-side-out orientation.     

Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism.

Organelles of the yeast Saccharomyces cerevisiae were isolated and analyzed for sterol composition and the activity of three enzymes involved in sterol metabolism. The plasma membrane and secretory vesicles, the fractions with the highest sterol contents, contain ergosterol as the major sterol. In other subcellular membranes, which exhibit lower sterol contents, intermediates of the sterol biosynthetic pathway were found at higher percentages. Lipid particles contain, in addition to ergosterol, large amounts of zymosterol, fecosterol, and episterol. These sterols are present esterified with long-chain fatty acids in this subcellular compartment, which also harbors practically all of the triacylglycerols present in the cell but very little phospholipids and proteins. Sterol delta 24-methyltransferase, an enzyme that catalyzes one of the late steps in sterol biosynthesis, was localized almost exclusively in lipid particles. Steryl ester formation is a microsomal process, whereas steryl ester hydrolysis occurs in the plasma membrane and in secretory vesicles. The fact that synthesis, storage, and hydrolysis of steryl esters occur in different subcellular compartments gives rise to the view that ergosteryl esters of lipid particles might serve as intermediates for the supply of ergosterol from internal membranes to the plasma membrane.     

Metabolism of Thiamine Triphosphate in Rat Brain: Correlation with Chloride Permeability

Abstract: Our results show that a net synthesis of thiamine triphosphate (TTP) can be demonstrated in vitro using rat brain extracts. The total homogenate was preincubated with thiamine or its diphosphate derivative (TDP), centrifuged, and washed twice. With TDP (1 m M ) as substrate, a 10-fold increase in TTP content was observed in this fraction (nuclear fraction, membrane vesicles). A smaller, but significant, increase was observed in the P₂ fraction (mitochondrial/synaptosomal fraction). In view of the low TTP content of our fractions, it was carefully assessed that authentic TTP was being formed. Incorporation of radioactivity from [β-³²P]TDP and [γ-³²P]ATP in TTP suggests that these two compounds are its precursors. Furthermore, TTP synthesis was inhibited by ADP and relatively low concentrations of Zn²⁺. These results suggest that TTP synthesis is catalyzed by an ATP:TDP transphosphorylase rather than by the cytoplasmic adenylate kinase that may be present in the vesicles. After osmotic lysis of the vesicles at alkaline pH, TTP was recovered in protein-bound form. Concomitantly, a soluble thiamine triphosphatase, with alkaline pH optimum, was also released from the vesicles. No net synthesis could be obtained in the cytosolic fraction or in detergent-solubilized systems. Like TTP synthesis, chloride permeability of the vesicles was increased when the homogenate had been incubated with thiamine and particularly with TDP. Our results suggest a regulatory role of TTP on chloride permeability, but the target remains to be characterized.     

Separation of inner and outer membranes of Rhodopseudomonas spheroides.

The separation of inner and outer membrane of Rhodopseudomonas spheroides has been achieved by means of sucrose density gradient (20%, 40%, 60%, w/w) centrifugation. The upper fraction of the gradient, with a specific density 1.181 (g/cm3), is high in cytochrome and succinate dehydrogenase activities, low in lipopolysaccharides and it is designated the inner membrane fraction. The bottom fraction of the gradient, with a specific density 1.240, is high in lipopolysaccharide and contains neither cytochrome nor succinate dehydrogenase activities. This fraction is the cell wall or outer membrane fraction. The intermediate band on the gradient is an unseparated fraction of inner and outer membrane fragments. This fraction has a specific denisty of 1.211 and represents less than 3% of total crude envelope. Thin sections of the vesicles of the inner membrane fraction and those of outer membrane provide morphological evidence for the identity of the individual membrane fractions. At least 22 protein bands are resolved by employing sodium dodecyl sulfate slab gel electrophoresis. Six bands are present only in the inner membrane and two bands are found exclusively in the outer membrane. Most of the remaining polypeptides are present in greater amounts in the inner membrane relative to the outer membrane fractions.