Isolation and characterization of two distinct membrane fractions from the Gram-positive nucleotide producer Brevibacterium ammoniagenes ATCC 6872

Abstract Bouyant density gradient centifugation of 'crude membranes' of the coryneform bacterium Brevibacterium ammoniagenes yielded two distinct membrane fractions which differed significantly in equilibrium densities (1.15 and 1.18 g/cm³), NADH dehydrogenase activity (0.29 and 0.09 μmol·min⁻¹·mg⁻¹), protein composition and association of ribosomes. As determined by one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) proteins unique to either the free membrane (FM) fraction or the denser ribosome-containing complexed membrane (CM) fraction were identified.     

Localization of Ribosome Formation in Escherichia Coli

Abstract

A number of ribonucleoprotein fractions have been isolated from Escherichia coli K 12, one of which is more strongly bound to the cell membrane than the rest and can be detached only by deoxycholate treatment. Several properties have been analyzed. The following are common to all fractions: Sedimentation characteristics of sub-units; Sedimentation constant, nucleotide composition and capacity to hybridize with DNA of RNA; electrophoretic behaviour of proteins. Other properties differ in the various fractions: cell membrane fractions have higher RNA/protein ratio, are more sensitive to RNAase and dissociate more easily into two subunits. By examining the rate of incorporation of [2-¹⁴C]-uracil into the various fractions, it has been found that cell membrane-bound ribonucleoprotein fractions contain a higher proportion of newly formed rRNA and part of this early-labeled rRNA is contained in particles sedimenting as ribosome precursors. Moreover, by isolating ribonucleoprotein fractions from cells grown in the presence of chloramphenicol, cell membrane-bound riconucleoprotein fraction is richer in CM particles. All these results lead to the conclusion that this fraction contains a higher proportion of ribosome precursors. The meaning of these results is discussed.     

A comparative study of the molecular structures of the plasma membranes and the smooth and the rough endoplasmic-reticulum membranes from rat liver

Plasma-membrane as well as smooth-, rough- and degranulated-endoplasmic-reticulum-membrane fractions were isolated from the microsomal pellet of rat liver. The purity of these fractions, as determined by marker-enzyme activities, electron microscopy, cholesterol content and RNA content, was found to be adequate for a comparative structural study. Major differences in lipid and protein composition were found to exist between the plasma membrane and the endoplasmic reticulum, but not between the smooth and the rough fractions of the endoplasmic reticulum. Differences in the location of membrane protein thiol groups and the mobility of the membrane phospholipids were observed between the plasma membranes and the endoplasmic reticulum, and these could be explained by differences in protein and lipid composition. However, by employing fluorescence and spin-labelling techniques structural changes were also observed between the smooth and the rough endoplasmic-reticulum fractions. These results suggest that the structural heterogeneity existing between the two latter membrane fractions occurs near or on their membrane surfaces and is not due to the greater number of ribosomes bound to the rough endoplasmic-reticulum fraction.     

A mitochondrial protein from Neurospora crassa detected both on ribosomes and in membrane fractions. Analysis of the gene, the message, and the protein

We have isolated clones representing at least three nuclear genes for mitochondrial ribosomal proteins from Neurospora crassa by screening a lambda gt11 cDNA library with an antiserum against a mixture of these proteins. The cDNA and genomic DNA sequence for one of these genes, mrp-3, was determined. The MRP3 protein was purified by immune-affinity chromatography, using a monoclonal antibody probe, and subjected to amino acid sequence analysis to identify the mature amino terminus and a prospective mitochondrial-targeting presequence. MRP3 was identified as the largest, least basic protein detected from the small subunit of ribosomes which had been salt-washed and fractionated on sucrose gradients. However, the mRNA and protein products of mrp-3 were found to be present in excess over those of other N. crassa mitoribosomal protein genes. Using a solution hybridization/S1 nuclease assay, we found three-fold- more mRNA for mrp-3 than for another mito-ribosomal protein gene. In addition, a 30- to 50-fold excess of non-ribosomal MRP3 protein was discovered. The additional protein was localized in mitochondrial membrane fractions; none was detected in matrix fractions after removal of the ribosomes. An immunologically related protein was detected in ribosome and membrane fractions of mitochondria from Saccharomyces cerevisiae. The functional significance of this dual localization remains an enigma.     

MEMBRANE PROTEINS OF RAT BRAIN: COMPOSITIONAL CHANGES DURING POSTNATAL DEVELOPMENT

Abstract— Membrane fractions from forebrain of rat were isolated at ages ranging from 5 to 93 days. Among these fractions were total membranes, three fractions isolated by density gradient centrifugation, and three subfractions which consisted of purified myelin and of two supernatant fractions. All membrane fractions showed an increase in protein content during the first postnatal month; however, only the myelin fraction and one of its supernatant fractions showed a prolonged accumulation. Myelin protein increased continually from 0.17 mg/g brain at 15 days to 8.3 mg/g brain at 93 days.
All fractions were analysed for protein composition by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Characteristic changes in protein composition were noted during postnatal development, most of which were pronounced up to the age of 20 days. Among others was a decrease in histones as compared to other proteins, with a concomitant shift in preponderance from the slow- to the fast-migrating histone band. In parallel, other proteins of high molecular weight became more prominent. No myelin could be isolated at 5 and 10 days. The deposition of myelin proteins was parallelled by the appearance of the Wolfgram protein which points to a close correlation of the Wolfgram protein to the process of myelination.     

Characterization of an RNA granule from developing brain

In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding beta-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.     

The separation of rat liver endoplasmic reticulum membrane proteins by two dimensional polyacrylamide gel electrophoresis

The separation of rat liver endoplasmic reticulum membrane proteins by two dimensional polyacrylamide gel electrophoresis is described. By this method, the proteins of the rough membrane ribosomes could be separated from the other rough membrane proteins. Both rough and smooth membrane fractions contain at least 30 defined membranal proteins. The electrophoretic patterns of rough and smooth membrane proteins are clearly different.     

Metalloproteinase activity is the sole factor responsible for the growth-promoting effect of conditioned medium in Trichoplusia ni insect cell cultures

Conditioned medium (CM) taken from a serum-free culture of Trichoplusia ni (BTI-Tn-5B1-4, High Five) cells on days 2 and 3, shortened the lagphase and increased the maximum cell density when added to T. ni cultures with low-inoculum cell density. Gel filtration fractions of CM, eluting at around 45kDa, stimulated cell proliferation even better than CM. A protein in the gel filtration fraction was identified by N-terminal amino acid sequencing as a proteinase, related to a snake venom metalloproteinase. Casein zymography showed, multiple metalloproteinase bands between 48 and 25kDa, as well as precursor forms above 48kDa. Metalloproteinase bands below the main band at 48kDa were autocatalytic degradation products. Metalloproteinase activity was the sole factor responsible for the growth stimulating effect of CM as shown by using the specific metalloproteinase inhibitor dl-thiorphan. Metalloproteinases have recently been shown to release growth factors from sequestering extracellular proteins. We propose that the metalloproteinase is involved in autocrine regulation of T. ni proliferation in serum-free media. In addition, a gel filtration fraction of CM, eluting at about 10kDa, inhibited cell growth. Apart from a lysozyme precursor protein and a cyclophilin-like protein, a kazal-type proteinase inhibitor could be identified in this fraction.     

Characteristics of the fractional composition of membranes of membrane-bound ribosomes

Electrophoresis in PAAG was used to study the fractional composition of proteins from free and membrane-bound ribosomes isolated from the rat liver. The membrane-bound ribosomes are shown to differ in the protein composition from the free ones in the presence of two fractions with a molecular weight of 35 and 50 kDa which are localized in a large subunit of the organelle. The possible role of these fractions in ribosome-membrane interaction is discussed.     

Qualitative and quantitative profiling of the bovine milk fat globule membrane proteome

Milk is a biological fluid of unique quality and complexity. It has co-evolved with mammals and mankind to nourish offspring and contains macro- and micronutrients for growth and development of the newborn. The milk fat globule membrane (MFGM) represents an important milk fraction, which is rich in bioactive proteins. In order to better understand functionality of milk fractions and, thereby, enhance the benefits of milk products, detailed qualitative and quantitative protein knowledge of fractions such as MFGM is required.We report the qualitative and quantitative profiling of two MFGM-enriched milk fractions, a whey protein concentrate (WPC) and a buttermilk protein concentrate (BMP), as derived from three different analytical workflows. First, an LC-MS/MS-based shotgun approach revealed 244 protein identities in WPC and 133 in BMP, respectively, and provided an extensive characterisation of the protein content in those two fractions. Second, label-free profiling resulted in rapid and efficient semi-quantitative comparison and yielded valuable protein fingerprints. Third, absolute quantification of selected MFGM proteins was achieved by stable isotope dilution (SID)-MS, in combination with multiple reaction monitoring (MRM) detection. In summary, we provide new information on composition, quantity and possible health benefits of two MFGM-enriched milk fractions highly valuable for future nutritional applications.     

Interaction of mammalian mitochondrial ribosomes with the inner membrane

All of the products of mitochondrial protein biosynthesis in animals are hydrophobic proteins that are localized in the inner membrane. Hence, it is possible that the synthesis of these proteins could occur on ribosomes associated with the inner membrane. To examine this possibility, inner membrane and matrix fractions of bovine mitochondria were examined for the presence of ribosomes using probes for the rRNAs. Between 40 and 50% of the ribosomes were found to fractionate with the inner membrane. About half of the ribosomes associated with the inner membrane could be released by high salt treatment, indicating that they interact with the membrane largely through electrostatic forces. No release of the ribosome was observed upon treatment with puromycin, suggesting that the association observed is not due to insertion of a nascent polypeptide chain into the membrane. A fraction of the ribosomes remained with residual portions of the membranes that cannot be solubilized in the presence of Triton X-100. These ribosomes may be associated with large oligomeric complexes in the membrane.     

Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity.

A membrane protein fraction showing affinity for ribosomes was isolated from rat liver microsomes (microsomal fractions) in association with ribosomes by treatment of the microsomes with Emulgen 913 and then solubilized from the ribosomes with sodium deoxycholate. This protein fraction was separated into two fractions, glycoproteins, including ribophorins I and II, and non-glycoproteins, virtually free from ribophorins I and II, on concanavalin A-Sepharose columns. The two fractions were each reconstituted into liposomes to determine their ribosome-binding activities. The specific binding activity of the non-glycoprotein fraction was approx. 2.3-fold higher than that of the glycoprotein fraction. The recovery of ribosome-binding capacity of the two fractions was about 85% of the total binding capacity of the material applied to a concanavalin A-Sepharose column, and about 90% of it was found in the non-glycoprotein fraction. The affinity constants of the ribosomes for the reconstituted liposomes were somewhat higher than those for stripped rough microsomes. The mode of ribosome binding to the reconstituted liposomes was very similar to that to the stripped rough microsomes, in its sensitivity to proteolytic enzymes and its strong inhibition by increasing KCl concentration. These results support the idea that ribosome binding to rat liver microsomes is not directly mediated by ribophorins I and II, but that another unidentified membrane protein(s) plays a role in ribosome binding.     

Aspects of the protein and the lipid composition of myelinoid marchi-positive bodies from mammalian spinal cord

The fraction floating on 0.32 M sucrose was isolated from normal mammalian spinal cord and analyzed with regard to protein and lipid composition. Comparisons were made with the myelin fraction isolated from the same spinal cord. A close relationship between the two fractions was indicated by a similar protein banding on SDS-polyacrylamide gel electrophoresis. The relative amounts of various proteins however were different and some high molecular weight proteins appeared unique to the floating fraction. The phospho- and galactolipid patterns, as revealed by thin-layer chromatography, were similar in the floating and the myelin fractions. The proportion of hydrophobic lipids, such as sterols and isoprenyl derivatives, was higher in the floating fraction. Bands co-migrating with cholesterol esters were detected only in the floating fraction from guinea pigs. Marchi-positive material of possible paranodal origin is enriched in the floating fraction. The present findings of a biochemical composition of the floating fraction closely resembling that of myelin is in line with the view that myelin turnover includes a step of degradation localized to the paranodal regions.     

Stoichiometric analysis of barley plastid ribosomal proteins

We analyzed the protein composition of plastid 70S ribosomes isolated from the stromal fractions of barley plastids by the radical-free and highly reducing method of two dimensional polyacrylamide gel electrophoresis (RFHR 2D-PAGE). Intactness of the ribosomes was confirmed by the poly(U)-directed phenylalanine polymerization activity and by the reassociation capacity of the subunits into 70S ribosomes. The small and large ribosomal subunits were composed of 23 and 36 proteins, respectively. In addition, one acidic protein associated with ribosomes in low salt buffer but released in high salt buffer was found. The plastid ribosomes contained relatively larger numbers of acidic proteins than prokaryotic ribosomes. Stoichiometric analysis revealed the presence of several ribosomal proteins in low copy numbers, indicating that the ribosomes of plastids were heterogeneous. We also investigated the protein composition of plastid ribosomes from greening barley leaves and found that it did not change during greening.     

Ribosomal protein gene sequence changes in erythromycin-resistant mutants of Escherichia coli.

The genes for ribosomal proteins L4 and L22 from two erythromycin-resistant mutants of Escherichia coli have been isolated and sequenced. In the L4 mutant, an A-to-G transition in codon 63 predicted a Lys-to-Glu change in the protein. In the L22 strain, a 9-bp deletion removed codons 82 to 84, eliminating the sequence Met-Lys-Arg from the protein. Consistent with these DNA changes, in comparison with wild-type proteins, both mutant proteins had reduced first-dimension mobilities in two-dimensional polyacrylamide gels. Complementation of each mutation by a wild-type gene on a plasmid vector resulted in increased erythromycin sensitivity in the partial-diploid strains. The fraction of ribosomes containing the mutant form of the protein was increased by growth in the presence of erythromycin. Erythromycin binding was increased by the fraction of wild-type protein present in the ribosome population. The strain with the L4 mutation was found to be cold sensitive for growth at 20 degrees C, and 50S-subunit assembly was impaired at this temperature. The mutated sequences are highly conserved in the corresponding proteins from a number of species. The results indicate the participation of these proteins in the interaction of erythromycin with the ribosome.     

Endoplasmic reticulum-bound ribosomes reside in stable association with the translocon following termination of protein synthesis

In current views, translation-coupled ribosome binding to the endoplasmic reticulum (ER) membrane is transient, with association occurring via the signal recognition particle pathway and dissociation occurring upon the termination of protein synthesis. Recent studies indicate, however, that ribosomal subunits remain membrane-bound following the termination of protein synthesis. To define the mechanism of post-termination ribosome association with the ER membrane, membrane-bound ribosomes were detergent-solubilized from tissue culture cells at different stages of the protein synthesis cycle, and the composition of the ribosome-associated membrane protein fraction was determined. We report that ribosomes reside in stable association with the Sec61alpha-translocon following the termination stage of protein synthesis. Additionally, in vitro experiments revealed that solubilized, gradient-purified ribosome-translocon complexes were able to initiate the translation of secretory and cytosolic proteins and were functional in assays of signal sequence recognition. Using this experimental system, synthesis of signal sequence-bearing polypeptides yielded a tight ribosome-translocon junction; synthesis of nascent polypeptides lacking a signal sequence resulted in a disruption of this junction. On the basis of these data, we propose that in situ, ribosomes reside in association with the translocon throughout the cycle of protein synthesis, with membrane release occurring upon translation of proteins lacking topogenic signals.     

Studies on peroxisomal membranes

The phospholipid/protein ratios of rat liver peroxisomes, mitochondria and microsomes were determined and found to be 257 +/- 26, 232 +/- 20 and 575 +/- 20 nmol.mg-1, respectively. After correction for the loss of soluble protein, a peroxisomal ratio of 153 nmol.mg-1 was calculated. Organelle fractions were treated with sodium carbonate, whereafter membrane fragments containing integral membrane proteins were pelleted. For the membrane fractions of peroxisomes, mitochondria and microsomes phospholipid/protein ratios of 1054 +/- 103, 1180 +/- 90 and 1050 +/- 50 nmol.mg-1 were found, whereas 26 +/- 2, 20 +/- 2 and 49 +/- 2% of the organelle protein was recovered in these membrane fractions, respectively. The phospholipid composition of the different organelle fractions were determined, but no large differences were obtained, except for cardiolipin that was found only in the mitochondrial fraction. After sodium carbonate treatment virtually all enzymatic activity of the enzymes tested was lost. Therefore Triton X-114 phase separation was used to obtain the peroxisomal membrane components. In this fraction 42.9 +/- 3.5% of the protein and 90.2 +/- 3.7% of the phospholipid was found. Enzymatic activity of two integral membrane proteins was recovered for over 90% in the membrane fraction, whereas activity of two matrix proteins was mainly found in the soluble fraction. Urate oxidase, the peroxisomal core protein, behaved differently and was recovered mainly with the membrane components. Recoveries of enzymatic activities after the Triton X-114 phase separation varied from 45 to 116%, and together with the good separation that was obtained between soluble proteins and integral membrane proteins this method provides a useful alternative for the isolation of membrane components.     

Comprehensive yeast proteome analysis using a capillary isoelectric focusing-based multidimensional separation platform coupled with ESI-MS/MS

As demonstrated in this study, a CIEF-based multidimensional separation platform not only is compatible with the detergent-based membrane protein preparation protocol, but also achieves both the largest yeast membrane proteome coverage and the most comprehensive analysis of the yeast proteome to date. By using a 1% false discovery rate for total peptide identifications, a total of 2513 distinct yeast proteins are identified from the SDS-solubilized fraction with an average of 5.4 peptides leading to each protein identification. Among proteins identified from the SDS-solubilized fraction, 407 proteins are predicted to contain at least two or more transmembrane domains using TMHMM (www.cbs.dtu.dk/services/TMHMM-2.0/), corresponding to 46% yeast membrane proteome coverage. Only four additional membrane proteins are identified in the soluble and urea-solubilized fractions, affirming the utility of SDS extraction for enriching the membrane proteome. By combining proteome results obtained from the soluble, urea-solubilized, and SDS-solubilized fractions, a single yeast proteome analysis yields the identification of 3632 distinct yeast proteins, corresponding to 55% theoretical yeast proteome coverage or 70% of proteins predicted to be expressed during log-phase growth in rich media.     

Detection of lateral heterogeneity in the cytoplasmic membrane of<Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Lateral heterogeneity in the cytoplasmic membrane of Bacillus subtilis was found by using density gradient centrifugation. Crude membranes (CM) present in the whole cell lysate were separated into three fractions of increasing density (F, CI, CII). Substantial difference exists in the amount of protein recovered from these fractions, the relative ratio being 15 : 35 : 50. The qualitative protein composition (by SDS-PAGE) of the fractions varies markedly as well. The lipid components extracted from the fractions are also distributed in different proportions, viz. 40 : 40 : 20. The spectrum of fatty acids (FA), detected in lipids of F fraction and analyzed by GC-MS exhibits the same profile as that found in CM; in contrast, fractions CI and CII undergo extensive FA reconstruction. Thermotropic behavior of fractions measured by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene indicates significant variations of microviscosity (r(s)) within the F, CI and CII fractions. The protein-to-lipid ratio plays evidently a key role in affecting the physical state of the cytoplasmic membrane. Microdomains of different density coexist in the membrane and exhibit heterogeneity in both chemical composition and "physical state"; the increased de novo synthesis of FA induced by the cold exclusively in fractions CI and CII indicates correlation with an altered physiological state of bacterial metabolism.     

Tyrosine phosphorylation of membrane proteins mediates cellular invasion by transformed cells

Tyrosine phosphorylation of membrane-associated proteins is involved at two distinct sites of contact between cells and the extracellular matrix: adhesion plaques (cell adhesion and de-adhesion) and invadopodia (invasion into the extracellular matrix). Adhesion plaques from chicken embryonic fibroblasts or from cells transformed by Rous sarcoma virus contain low levels of tyrosine-phosphorylated proteins (YPPs) which were below the level of detection in 0.5-microns thin, frozen sections. In contrast, intense localization of YPPs was observed at invadopodia of transformed cells at sites of degradation and invasion into the fibronectin-coated gelatin substratum, but not in membrane extensions free of contact with the extracellular matrix. Local extracellular matrix degradation and formation of invadopodia were blocked by genistein, an inhibitor of tyrosine-specific kinases, but cells remained attached to the substratum and retained their free-membrane extensions. Invadopodia reduced or lost YPP labeling after treatment of the cells with genistein, but adhesion plaques retained YPP labeling. The plasma membrane contact fractions of normal and transformed cells have been isolated form cells grown on gelatin cross-linked substratum using a novel fractionation scheme, and analyzed by immunoblotting. Four major YPPs (150, 130, 81, and 77 kD) characterize invadopodial membranes in contact with the matrix, and are probably responsible for the intense YPP labeling associated with invadopodia extending into sites of matrix degradation. YPP150 may be an invadopodal-specific YPP since it is approximately 3.6-fold enriched in the invasive contact fraction relative to the cell body fraction and is not observed in normal contacts. YPP130 is enriched in transformed cell contacts but may also be present in normal contacts. The two major YPPs of normal contacts (130 and 71 kD) are much lower in abundance than the major tyrosine-phosphorylated bands associated with invadopodial membranes, and likely represent major adhesion plaque YPPs. YPP150, paxillin, and tensin appear to be enriched in the cell contact fractions containing adhesion plaques and invadopodia relative to the cell body fraction, but are also present in the soluble supernate fraction. However, vinculin, talin, and alpha-actinin that are localized at invadopodia, are equally concentrated in cell bodies and cell contacts as is the membrane-adhesion receptor beta 1 integrin. Thus, tyrosine phosphorylation of the membrane-bound proteins may contribute to the cytoskeletal and plasma membrane events leading to the formation and function of invadopodia that contact and proteolytically degrade the extracellular matrix; we have identified several candidate YPPs that may participate in the regulation of these processes.