Subcellular Fractionation Methods Research Articles

Quantitative RT-PCR Analysis of Influenza Virus Endocytic Escape.

Although several virus families are internalized into their host cells by direct fusion of the viral envelope with the plasma membrane, most viruses, for example, influenza virus, make use of endocytic pathways for productive entry and infection. After endocytosis, the influenza virus escapes from the endocytic compartment to the cytosol. The distribution of the incoming influenza virus could be traced by detection of the viral RNA in the distinct cellular compartments, including endosome, cytosol, and nucleus. To accomplish this work, we developed a subcellular fractionation method based on density gradient ultracentrifugation and detected the viral RNA using quantitative reverse transcription-polymerase chain reaction analysis. This chapter is devoted to the practical methods and precautions for studying endocytic traffic of virus as well as host cellular factors affecting viral endocytosis.

AMPA receptor translocation and phosphorylation are induced by transcranial direct current stimulation in rats.

Over the last decade, the interest in transcranial direct current stimulation (tDCS) has continued to increase, along with consideration of how it affects neuroplasticity mechanisms in the brain. Both human and animal studies have demonstrated numerous benefits and, although its application has increased, the neurophysiological mechanisms underlying tDCS' beneficial effects remain largely unknown. Recent studies have shown that long-term potentiation (LTP) increases following tDCS. In this work, we utilized a rodent model of tDCS to directly assess changes in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, a critical protein for enhancing synaptic transmission. Animals were subjected to 250 μA of direct current (DC) stimulation for 30 min with immediate tissue collection. Translocation and phosphorylation of AMPA receptors were examined using protein immunoblot analysis following a subcellular fractionation method. Our findings show that a single application of in vivo tDCS can affect both the translocation and phosphorylation of AMPA receptors in the hippocampus while increasing AMPA receptor phosphorylation in the hypothalamus. In the hippocampus, tDCS increased AMPA translocation to the synapse and increased the phosphorylation of the S831 site on GluA1. In the hypothalamus, no statistically significant changes were observed in AMPA translocation while an increase in the phosphorylation of the S831 site was observed. No changes in the phosphorylation of GluA1 at the S845 site were detected in either brain region. In sum, our findings identify specific AMPA receptor changes induced by tDCS, thereby providing further details on the mechanisms by which tDCS could affect the establishment of LTP and modulate neuroplasticity.

Rapid and facile quantitation of polyplex endocytic trafficking

Design of safe and effective synthetic nucleic acid delivery vectors such as polycation/DNA or polycation/siRNA complexes (polyplexes) will be facilitated by quantitative understanding of the mechanisms by which such materials escort cargo from the cell surface to the nucleus. In particular, the mechanisms of cellular internalization by various endocytosis pathways and subsequent endocytic vesicle trafficking have been shown to strongly affect nucleic acid delivery efficiency. Fluorescence microscopy and subcellular fractionation methods are commonly employed to follow intracellular trafficking of biomolecules and nanoparticulate delivery systems such as polyplexes. However, it is difficult to obtain quantitative data from microscopy and subcellular fractionation is experimentally difficult and low throughput. We have developed a method for quantifying the transport of polyplexes through important endocytic vesicles. The method is based on polymerization of 3,3′-diaminobenzidine by endocytosed horseradish peroxidase, causing an increase in the vesicle density, resistance to being solubilized by detergent and quenching of fluorophores within the vesicles, which makes them easy to separate and quantify. Using this method in HeLa cells, we have observed polyethylenimine/siRNA polyplexes initially appearing in early endosomes and rapidly moving to other compartments within 30min post-transfection. At the same time, we observed the kinetics of accumulation of the polyplexes in lysosomes at a similar rate. The results from the new method are consistent with similar measurements by confocal fluorescence microscopy and subcellular fractionation of endocytic vesicles on a Percoll gradient. The relative ease of this new method will aid investigation of gene delivery mechanisms by providing the means to rapidly quantify endocytic trafficking of polyplexes and other vectors.

Purification of Synaptosome Populations Using Fluorescence-Activated Synaptosome Sorting.

For several decades, neurobiologists have used subcellular fractionation methods to analyze the molecular structure and some functional features of the cells in the central nervous system. Indeed, brain tissue contains a complex intermingled network of neuronal, glial, and vascular cells. To reduce this complexity biochemists have optimized fractionation protocols that enrich in specific compartments such as synapses (called "synaptosomes") and synaptic vesicles, for example. However, recently, these approaches suffered from a lack of specificity and purity. In a recent effort, we extended the conventional synaptosome preparation to purify fluorescent synaptosomes on a cell sorter. We could prove that our method allows for the steep enrichment in fluorescent excitatory VGLUT1venus synaptosomes containing the presynaptic element and the tip of the post-synaptic element and a strong depletion in neuronal and glial contaminants. Here, we propose a detailed procedure for the implementation of Fluorescence Activated Synaptosome Sorting.

Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization

Two different putative galactokinase genes, found in the genome database of Trypanosoma cruzi were cloned and sequenced. Expression of the genes in Escherichia coli resulted for TcGALK-1 in the synthesis of a soluble and active enzyme, and in the case of TcGALK-2 gene a less soluble protein, with predicted molecular masses of 51.9kDa and 51.3kDa, respectively. The Km values determined for the recombinant proteins were for galactose 0.108mM (TcGALK-1) and 0.091mM (TcGALK-2) and for ATP 0.36mM (TcGALK-1) and 0.1mM (TcGALK-2). Substrate inhibition by ATP (Ki 0.414mM) was only observed for TcGALK-2. Gel-filtration chromatography showed that natural TcGALKs and recombinant TcGALK-1 are monomeric. In agreement with the possession of a type-1 peroxisome-targeting signal by both TcGALKs, they were found to be present inside glycosomes using two different methods of subcellular fractionation in conjunction with mass spectrometry. Both genes are expressed in epimastigote and trypomastigote stages since the respective proteins were immunodetected by western blotting. The T. cruzi galactokinases present their highest (52–47%) sequence identity with their counterpart from Leishmania spp., followed by prokaryotic galactokinases such as those from E. coli and Lactococcus lactis (26–23%). In a phylogenetic analysis, the trypanosomatid galactokinases form a separate cluster, showing an affiliation with bacteria. Epimastigotes of T. cruzi can grow in glucose-depleted LIT-medium supplemented with 20mM of galactose, suggesting that this hexose, upon phosphorylation by a TcGALK, could be used in the synthesis of UDP-galactose and also as a possible carbon and energy source.

Subcellular fractionation method to study endosomal trafficking of Kaposi's sarcoma-associated herpesvirus.

BackgroundVirus entry involves multiple steps and is a highly orchestrated process on which successful infection collectively depends. Entry processes are commonly analyzed by monitoring internalized virus particles via Western blotting, polymerase chain reaction, and imaging techniques that allow scientist to track the intracellular location of the pathogen. Such studies have provided abundant direct evidence on how viruses interact with receptor molecules on the cell surface, induce cell signaling at the point of initial contact with the cell to facilitate internalization, and exploit existing endocytic mechanisms of the cell for their ultimate infectious agenda. However, there is dearth of knowledge in regards to trafficking of a virus via endosomes. Herein, we describe an optimized laboratory procedure to isolate individual organelles during different stages of endocytosis by performing subcellular fractionation. This methodology is established using Kaposi’s sarcoma-associated herpesvirus (KSHV) infection of human foreskin fibroblast (HFF) cells as a model. With KSHV and other herpesviruses alike, envelope glycoproteins have been widely reported to physically engage target cell surface receptors, such as integrins, in interactions leading to entry and subsequent infection.ResultsSubcellular fractionation was used to isolate early and late endosomes (EEs and LEs) by performing a series of centrifugations steps. Specifically, a centrifugation step post-homogenization was utilized to obtain the post-nuclear supernatant containing intact intracellular organelles in suspension. Successive fractionation via sucrose density gradient centrifugation was performed to isolate specific organelles including EEs and LEs. Intracellular KSHV trafficking was directly traced in the isolated endosomal fractions. Additionally, the subcellular fractionation approach demonstrates a key role for integrins in the endosomal trafficking of KSHV. The results obtained from fractionation studies corroborated those obtained by traditional imaging studies.ConclusionsThis study is the first of its kind to employ a sucrose flotation gradient assay to map intracellular KSHV trafficking in HFF cells. We are confident that such an approach will serve as a powerful tool to directly study intracellular trafficking of a virus, signaling events occurring on endosomal membranes, and dynamics of molecular events within endosomes that are crucial for uncoating and virus escape into the cytosol.

Assessing PTEN Subcellular Localization.

PTEN subcellular localization is fundamental in the execution of the distinct PTEN biological activities, including not only its PI(3,4,5)P3 phosphatase activity when associated to membranes but also its subcellular compartment-specific interactions with regulatory and effector proteins, including those exerted in the nucleus. As a consequence, PTEN subcellular localization is tightly regulated in vivo by both intrinsic and extrinsic mechanisms. The plasma membrane/nucleus/cytoplasm partitioning of PTEN has been the focus of several studies, both from a mechanistic and from a disease-association point of view. Here, we summarize the current knowledge on PTEN plasma membrane/nucleus/cytoplasm distribution, and present subcellular fractionation, immunofluorescence, and immunohistochemical methods to study the distribution and shuttling of PTEN between these subcellular compartments.

Mercury tissue residue approach in Chironomus riparius: Involvement of toxicokinetics and comparison of subcellular fractionation methods

Along with the growing body of evidence that total internal concentration is not a good indicator of toxicity, the Critical Body Residue (CBR) approach recently evolved into the Tissue Residue Approach (TRA) which considers the biologically active portion of metal that is available to contribute to the toxicity at sites of toxic action. For that purpose, we examined total mercury (Hg) bioaccumulation and subcellular fractionation kinetics in fourth stage larvae of the midge Chironomus riparius during a four-day laboratory exposure to Hg-spiked sediments and water. The debris (including exoskeleton, gut contents and cellular debris), granule and organelle fractions accounted only for about 10% of the Hg taken up, whereas Hg concentrations in the entire cytosolic fraction rapidly increased to approach steady-state. Within this fraction, Hg compartmentalization to metallothionein-like proteins (MTLP) and heat-sensitive proteins (HSP), consisting mostly of enzymes, was assessed in a comparative manner by two methodologies based on heat-treatment and centrifugation (HT&C method) or size exclusion chromatography separation (SECS method). The low Hg recoveries obtained with the HT&C method prevented accurate analysis of the cytosolic Hg fractionation by this approach. According to the SECS methodology, the Hg-bound MTLP fraction increased linearly over the exposure duration and sequestered a third of the Hg flux entering the cytosol. In contrast, the HSP fraction progressively saturated leading to Hg excretion and physiological impairments. This work highlights several methodological and biological aspects to improve our understanding of Hg toxicological bioavailability in aquatic invertebrates.

An efficient two-step subcellular fractionation method for the enrichment of insulin granules from INS-1 cells.

Insulin is one of the key regulators for blood glucose homeostasis. More than 99% of insulin is secreted from the pancreatic β-cells. Within each β-cell, insulin is packaged and processed in insulin secretary granules (ISGs) before its exocytosis. Insulin secretion is a complicated but well-organized dynamic process that includes the budding of immature ISGs (iISGs) from the trans-Golgi network, iISG maturation, and mature ISG (mISG) fusion with plasma membrane. However, the molecular mechanisms involved in this process are largely unknown. It is therefore crucial to separate and enrich iISGs and mISGs before determining their distinct characteristics and protein contents. Here, we developed an efficient two-step subcellular fractionation method for the enrichment of iISGs and mISGs from INS-1 cells: OptiPrep gradient purification followed by Percoll solution purification. We demonstrated that by using this method, iISGs and mISGs can be successfully distinguished and enriched. This method can be easily adapted to investigate SGs in other cells or tissues, thereby providing a useful tool for elucidating the mechanisms of granule maturation and secretion.

Critical determinants of mitochondria-associated neutral sphingomyelinase (MA-nSMase) for mitochondrial localization

BackgroundA novel murine mitochondria-associated neutral sphingomyelinase (MA-nSMase) has been recently cloned and partially characterized. The subcellular localization of the enzyme was found to be predominant in mitochondria. In this work, the determinants of mitochondrial localization and its topology were investigated. MethodsMA-nSMase mutants lacking consecutive regions and fusion proteins of GFP with truncated MA-nSMase regions were constructed and expressed in MCF-7 cells. Its localization was analyzed using confocal microscopy and sub-cellular fractionation methods. The sub-mitochondrial localization of MA-nSMase was determined using protease protection assay on isolated mitochondria. ResultsThe results initially showed that a putative mitochondrial localization signal (MLS), homologous to an MLS in the zebra-fish mitochondrial SMase is not necessary for the mitochondrial localization of the murine MA-nSMase. Evidence is provided to the presence of two regions in MA-nSMase that are sufficient for mitochondrial localization: a signal sequence (amino acids 24–56) that is responsible for the mitochondrial localization and an additional ‘signal-anchor’ sequence (amino acids 77–99) that anchors the protein to the mitochondrial membrane. This protein is topologically located in the outer mitochondrial membrane where both the C and N-termini remain exposed to the cytosol. ConclusionsMA-nSMase is a membrane anchored protein with a MLS and a signal-anchor sequence at its N-terminal to localize it to the outer mitochondrial membrane. General significanceMitochondrial sphingolipids have been reported to play a critical role in cellular viability. This study opens a new window to investigate their cellular functions, and to define novel therapeutic targets.

Visualizing the microtubule-associated protein tau in the nucleus

Although tau is mainly known as an axonal microtubule-associated protein, many studies indicate that it is not restricted to this subcellular compartment. Assessing tau's subcellular distribution, however, is not trivial as is evident from transgenic mouse studies. When human tau is over-expressed, it can be immunohistochemically localized to axons and the somatodendritic domain, modeling what is found in neurodegenerative diseases such as Alzheimer's disease. Yet, in wild-type mice, despite its abundance, tau is difficult to visualize even in the axon. It is even more challenging to detect this protein in the nucleus, where tau has been proposed to protect DNA from damage. To establish a framework for future studies into tau's nuclear functions, we compared several methods to visualize endogenous nuclear tau in cell lines and mouse brain. While depending on the fixation and permeabilization protocol, we were able to detect nuclear tau in SH-SY5Y human neuroblastoma cells, we failed to do so in N2a murine neuroblastoma cells. As a second method we used subcellular fractionation of mouse tissue and found that in the nucleus tau is mainly present in a hypophosphorylated form. When either full-length or truncated human tau was expressed, both accumulated in the cytoplasm, but were also found in the nuclear fraction. Because subcellular fractionation methods have their limitations, we finally isolated nuclei to probe for nuclear tau and found that the nuclei were free of cytoplasmic contamination. Together our analysis identifies several protocols for detecting tau in the nucleus where it is found in a less phosphorylated form.

Chronic Copper Poisoning in Sheep: Liver Injury

Pathogenesis of liver injury in chronic copper poisoning of sheep was investigated using two experiments. Test sheep were dosed orally with a 0.2 % solution of CuSO4.5H2O at the rate of 10 ml/Kg body weight on five days of the week, until first day of the haemolytic crisis (HC). Liver samples were taken during the prehaemolytic period and on the first or second day of the HC, liver changes were evaluated using trace element, histochemical, subcellular fractionation, ultrastructural and morphometric methods. Copper concentrations in liver samples increased at a steady linear rate of 10.35 µg/g liver wet weight per day; uptake of copper among hepatocytes, among Kupffer cells and between zones of liver lobules was unequal. Individual hepatocytes became packed with copper loaded lysosomes and underwent degeneration and necrosis. Excess copper in liver cells was sequestered by the lysosomes leading to linear increases in volume density, numerical density and mean volume during the pre-haemolytic period. On the first day of HC, numerical density reduced, volume density remained unchanged and the mean volume increased. Linear increase in liver copper concentration indicates that copper absorption from the gut; excretion into bile and release into sinusoids by hepatocytes occur at linear steady rates. Thus, copper homeostasis in sheep is different to that of the humans and rats. In hepatocytes packed with copper loaded lysosomes, synthesis of new lysosomes reduced, causing decreased uptake of excess copper leading to accumulation of ionic copper in the cytoplasm, resulting in the degeneration and necrosis of the hepatocytes.

Investigation of Polyethylenimine/DNA Polyplex Transfection to Cultured Cells Using Radiolabeling and Subcellular Fractionation Methods

Quantitative analysis of the intracellular trafficking of nonviral vectors provides critical information that can guide the rational design of improved cationic systems for gene delivery. Subcellular fractionation methods, combined with radiolabeling, produce quantitative measurements of the intracellular trafficking of nonviral vectors and the therapeutic payload. In this work, differential and density-gradient centrifugation techniques were used to determine the intracellular distribution of radiolabeled 25 kDa branched polyethylenimine (bPEI)/plasmid DNA complexes ("polyplexes") in HeLa cells over time. By differential centrifugation, [(14)C]bPEI was found mostly in the lighter fractions whereas [(3)H]DNA was found mostly in the heavier fractions. A majority of the intracellular polymer (∼60%) and DNA (∼90%) were found in the nuclear fraction. Polymer and DNA also differed in their distribution to heavier and denser organelles (lysosomes, mitochondria) in density-gradient centrifugation studies. An unexpected finding from this study was that between 18 and 50% of the DNA applied to the cells became cell-associated (either with the cell membrane and/or internalized), while only 1-6% of the polymer did so, resulting in an effective N/P ratio of less than 1. These results suggest that a significant amount of cationic polymer is dissociated from the DNA cargo early on in the transfection process.

A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue

BackgroundWe describe a method for subcellular fractionation of mouse skeletal muscle, myoblast and myotubes to obtain relatively pure fractions of nuclear, cytosolic and mitochondrial compartments. Fractionation allows the analysis of a protein of interest (or other cellular component) based on its subcellular compartmental distribution and can also generate molecular information about the state of a cell and/or tissue and how the distribution of a protein may differ between different cellular compartments, tissues or cell types, in response to treatments or ageing.FindingsThe described method was specifically developed for skeletal muscle and proliferating/differentiated muscle cells. The purity of the different fractions, representing the cytoplasmic, mitochondrial and nuclear subcellular compartments was validated by western blot analysis of “house-keeper” marker proteins specific for each cellular compartment.ConclusionThis low cost method allowed the mitochondrial, cytoplasmic and nuclear subcellular compartments from the same starting muscle samples to be rapidly and simultaneously isolated with good purity and without the use of an ultracentrifuge. This method permits samples to be frozen at −80°C for future analysis and/or additional processing at a later date.

Abstract 1489: The role of bone marrow adipocyte-induced HO-1 expression in prostate tumor survival in bone

Abstract Prostate cancer (PCa) is the second leading cause of cancer deaths among men in the United States. Obesity, adipocyte-derived signals, and adipose tissue inflammation are major contributors to development of aggressive PCa, with higher recurrence and higher mortality rates. Bone is a primary site of metastasis of PCa and obese and overweight men have a three-fold higher risk of progression to metastatic disease compared to normal-weight men receiving the same treatment. Experimental and clinical evidence indicate that bone marrow adipocyte numbers increase with age and obesity and their increased presence affects homeostasis in the bone microenvironment. It is not, however, currently known how adipocyte-derived factors influence tumor growth and survival in bone. Heme oxygenase 1 (HO-1) is a stress-responsive protein induced by hypoxia and inflammation, and it has been shown to play a role in prostate carcinogenesis and cell survival. Its role in tumor cell aggressiveness and survival in response to adipocyte-derived signals has not been previously explored. Here, we investigated the effects of bone marrow adipocyte-derived factors on HO-1/IL-1α pathway in PCa cells. Our study was driven by hypothesis that adipocytes increase invasiveness and survival of PCa cells via upregulation of HO-1. We utilized Taqman RT PCR analyses and western blotting approaches to demonstrate that levels of HO-1 and its inducer IL-1α increase in PC3, DU145 and C42B prostate cancer cell lines in response to treatment with adipocyte conditioned media (Adipo CM). Immunofluorescence analyses and subcellular fractionation methods were used to demonstrate a translocation of HO-1 from the cytoplasm to nucleus upon exposure to Adipo CM. Invasiveness of PCa cells in response to adipocyte-derived factors in the absence and presence of IL-1α neutralizing antibody and HO-1 inhibitor Zinc protoporphyrin IX was assessed using Boyden chamber assays. Studies are currently underway to determine the significance of HO-1/IL-1α upregulation in tumor cells survival and response to chemotherapy. Collectively, the results of our studies demonstrate that adipocyte-induced upregulation of HO-1/IL-1α pathways may be playing a significant role in promoting prostate tumor aggressiveness and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1489. doi:1538-7445.AM2012-1489

Differential expression of membrane proteins helps Antarctic Pseudomonas syringae to acclimatize upon temperature variations

Antarctic bacteria are adapted to the extremely low temperature. The transcriptional and translational machineries of these bacteria are adapted to the sub-zero degrees of temperature. Studies directed towards identifying the changes in the protein profiles during changes in the growth temperatures of an Antarctic bacterium Pseudomonas syringae Lz4W may help in understanding the molecular basis of cold adaptation. In this study, subcellular fractionation methods of proteins were used for the enrichment and identification of proteins including low abundance proteins. The membrane proteins of the bacterium P. syringae Lz4W were prepared employing sucrose density gradient method. The proteins were separated through 2D gel-electrophoresis with the pH ranges 3-10, 4-7 and 5-8 using the detergent, amidosulfobetaine (ASB-14). The proteins separated on the 1D SDS PAGE and 2D gels were identified with the help of LC-ESI MS/MS and MALDI TOF TOF using bioinformatic programs MASCOT and SEQUEST. Since the genome sequence of P. syringae Lz4W is not available, the proteins are identified by using the genome database of the Pseudomonas sp. available at NCBI. The present studies focus on identifying temperature dependent expression of proteins by employing LC-MS/MS method and the functional significance of these proteins is discussed.

A Rice Transmembrane bZIP Transcription Factor, OsbZIP39, Regulates the Endoplasmic Reticulum Stress Response

The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways. These pathways are known as the ER stress response or the unfolded protein response. In this study, three rice basic leucine zipper (bZIP) transcription factors (OsbZIP39, OsbZIP50 and OsbZIP60) containing putative transmembrane domains (TMDs) in their C-terminal regions were identified as candidates of the ER stress sensor transducer. One of these proteins, OsbZIP39, was characterized in this study. OsbZIP39 was shown to associate with microsomes as a membrane-integrated protein using the subcellular fractionation method. When the full length and a truncated form of OsbZIP39 without the TMD (OsbZIP39ΔC) was fused to green fluorescent protein (GFP) and transfected into rice protoplasts, the proteins were identified in the cytoplasm and nucleus, respectively. This suggests that OsbZIP39 may be converted into a soluble truncated form by proteolytic cleavage and subsequently translocated to the nucleus. Expression of OsbZIP39ΔC clearly activated the binding protein 1 (BiP1) promoter in a rice protoplast transient assay. Overexpression of OsbZIP39ΔC in stable transgenic rice also led to the up-regulation of several ER stress response genes including BiP1 and OsbZIP50 in the absence of ER stress. However, in the OsbZIP39ΔC-overexpressing line, OsbZIP50 mRNA did not undergo IRE1 (inositol-requiring protein 1)-mediated cytoplasmic splicing that is required for its activation. These data indicate that OsbZIP39 may be directly involved in the regulation of several ER stress response genes.

Identification of Outer Membrane Proteins from an Antarctic Bacterium Pseudomonas syringae Lz4W

Subcellular fractionation of proteins is a preferred method of choice for detection and identification of proteins from complex mixtures such as bacterial cells. To characterize the membrane proteins of the Antarctic bacterium Pseudomonas syringae Lz4W, the membrane fractions were prepared using three different methods, namely Triton X-100 solubilization, sucrose density gradient, and carbonate extraction methods. The proteins were separated on one-dimensional polyacrylamide gels and analyzed using a combination of liquid chromatography-coupled electrospray ionization-MS. The membrane proteins that were prepared by carbonate extraction were separated on two-dimensional PAGE in different pI ranges using the detergent 2% amidosulfobetaine (ASB). The proteins were then subjected to matrix-assisted laser desorption ionization-time-of-flight/time-of-flight for analysis and identification. Because the genome sequence of P. syringae Lz4W is not known, the proteins were identified by using the relevant sequence databases of the Pseudomonas sp available at National Centre for Biotechnology Information (NCBI). The sequence identification of some tryptic peptides were validated by de novo sequencing and others by chemical modification and mass spectrometry. The peptide sequences of P. syringae Lz4W were then matched with the sequences of the peptides from different Pseudomonas sp. by similarity search of the proteins from different species using clustal W2 program. Thus by using a combination of the methods, we have been able to identify large number of proteins of this bacterial strain, which include most of the outer membrane proteins.

Regarding the Amazing Choreography of Clathrin Coats

The growth of contemporary cell biology is due in large part to technological advances. In the 1950s, electron micrographs of thin sections first provided unrivaled in situ views of the delicate intracellular architecture and fine structure of organelles, whereas new subcellular fractionation methods gave access to various biochemical components—especially proteins—enriched in different cellular fractions. A central tenet that emerged from these pioneering studies is that the intracellular biosynthetic and endocytic membrane systems of eukaryotic cells are functionally interconnected, and exchange of material between them often occurs in small (50–100 nm diameter), roughly spherical membranous transport vesicles. In electron micrographs, these vesicles are typically seen in close proximity to a membrane compartment and are frequently covered on their cytosolic face with a fuzzy proteinaceous coating. Subsequent technical advances facilitated further discovery and progress: genetic screens in model organisms and refinement of subcellular fractionation to facilitate cell-free reconstitution of transport reactions allowed the identification and purification of key regulatory and structural components. Persuasively, many of the genes discovered in genetic screens encoded the proteins purified biochemically. More recently, genome sequencing and proteomics efforts have bolstered the identification of sorting components, leading to long lists of evolutionarily conserved proteins that are involved in specific sorting operations at different membranes.

Biochemical localization of a protein involved in synthesis of Gluconacetobacter hansenii cellulose

Using subcellular fractionation and Western blot methods, we have shown that AcsD, one of the proteins encoded by the Acetobacter cellulose synthase (acs) operon, is localized in the periplasmic region of the cell. AcsD protein was heterologously expressed in Escherichia coli and purified using histidine tag affinity methods. The purified protein was used to obtain rabbit polyclonal antibodies. The purity of the subcellular fractions was assessed by marker enzyme assays.