16-BAC/SDS-PAGE Analysis of Membrane Proteins of Yeast Mitochondria Purified by Free Flow Electrophoresis

Endosomes are prelysosomal organelles that serve as an intracellular site for the sorting, distribution, and processing of receptors, ligands, fluid phase components, and membrane proteins internalized by endocytosis. Whereas the overall functions of endosomes are increasingly understood, little is known about endosome structure, composition, or biogenesis. In this paper, we describe a rapid procedure that permits analytical and preparative isolation of endosomes from a variety of tissue culture cells. The procedure relies on a combination of density gradient centrifugation and free flow electrophoresis. It yields a fraction of highly purified, functionally intact organelles. As markers for endosomes in Chinese hamster ovary cells, we used endocytosed horseradish peroxidase, FITC-conjugated dextran, and [35S]methionine-labeled Semliki Forest virus. Total postnuclear supernatants, crude microsomal pellets, or partially purified Golgi fractions were subjected to free flow electrophoresis. Endosomes and lysosomes migrated together as a single anodally deflected peak separated from most other organelles (plasma membrane, mitochondria, endoplasmic reticulum, and Golgi). The endosomes and lysosomes were then resolved by centrifugation in Percoll density gradients. Endosomes prepared in this way were enriched up to 70-fold relative to the initial homogenate and were still capable of ATP-dependent acidification. By electron microscopy, the isolated organelles were found to consist of electron lucent vacuoles and tubules, many of which could be shown to contain an endocytic tracer (e.g., horseradish peroxidase). SDS PAGE analysis of integral and peripheral membrane proteins (separated from each other by condensation in Triton X-114) revealed a unique and restricted subset of proteins when compared with lysosomes, the unshifted free flow electrophoresis peak, and total cell protein. Altogether, the purification procedure takes 5-6 h and yields amounts of endosomes (150-200 micrograms protein) sufficient for biochemical, immunological, and functional analysis.

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Detergent binding explains anomalous SDS-PAGE migration of membrane proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0813167106 ◽

2009 ◽

Vol 106 (6) ◽

pp. 1760-1765 ◽

Cited By ~ 469

Author(s):

Arianna Rath ◽

Mira Glibowicka ◽

Vincent G. Nadeau ◽

Gong Chen ◽

Charles M. Deber

Keyword(s):

Membrane Proteins ◽

Page Migration ◽

Sds Page

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Isolation of Monodisperse Nanodisc-Reconstituted Membrane Proteins Using Free Flow Electrophoresis

Analytical Chemistry ◽

10.1021/ac4000915 ◽

2013 ◽

Vol 85 (7) ◽

pp. 3497-3500 ◽

Cited By ~ 13

Author(s):

Bo Højen Justesen ◽

Tomas Laursen ◽

Gerhard Weber ◽

Anja Thoe Fuglsang ◽

Birger Lindberg Møller ◽

...

Keyword(s):

Membrane Proteins ◽

Free Flow

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Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. I. Isolation and characterization of membrane and content subfractions.

The Journal of Cell Biology ◽

10.1083/jcb.96.4.1030 ◽

1983 ◽

Vol 96 (4) ◽

pp. 1030-1039 ◽

Cited By ~ 6

Author(s):

W J Brown ◽

W A Shannon ◽

W J Snell

Keyword(s):

Membrane Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Protein Composition ◽

Granule Protein ◽

Isolation And Characterization ◽

Cytoplasmic Face ◽

Sds Page ◽

Granule Membrane ◽

Granule Membrane Proteins

The specific and azurophilic granules of rabbit polymorphonuclear heterophils (PMNs) have been isolated and fractionated into membrane and extractable subfractions. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) revealed several features of the protein composition of the two granules: (a) Whereas each type of granule had 40-60 proteins separable on one-dimensional gradient gels, few of the proteins were common to both granules. (b) The proteins of the extractable fractions (which comprised approximately 98% of the total granule protein) of each granule were distinct from the proteins of the membrane fractions (which comprised approximately 2% of the total granule protein). (c) The extractable proteins co-migrated with those collected from the medium of ionophore-treated, degranulating PMNs and therefore were defined as content proteins. These results were confirmed by radiolabeling studies. Lactoperoxidase-catalyzed iodination of intact granules did not label the content proteins but did label proteins that co-migrated with major granule membrane proteins. Moreover, disruption of the granules before iodination led to labeling of both content and membrane proteins. We conclude that the membranes of specific and azurophilic granules, which arise from different faces of the Golgi complex, are composed of unique sets of membrane proteins some of which are exposed on the cytoplasmic face of the granules.

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Establishing a Direct Role for the Bartonella bacilliformis Invasion-Associated Locus B (IalB) Protein in Human Erythrocyte Parasitism

Infection and Immunity ◽

10.1128/iai.69.7.4373-4381.2001 ◽

2001 ◽

Vol 69 (7) ◽

pp. 4373-4381 ◽

Cited By ~ 36

Author(s):

Sherry A. Coleman ◽

Michael F. Minnick

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Protein Localization ◽

Outer Membrane Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Buoyant Density ◽

Sodium Dodecyl ◽

Amino Acid Sequence Similarity ◽

Bartonella Bacilliformis ◽

Sds Page

ABSTRACT The invasion-associated locus A and B genes (ialAB) ofBartonella bacilliformis were previously shown to confer an erythrocyte-invasive phenotype upon Escherichia coli, indirectly implicating their role in virulence. We report the first direct demonstration of a role for ialB as a virulence factor in B. bacilliformis. The presence of a secretory signal sequence and amino acid sequence similarity to two known outer membrane proteins involved in virulence suggested that IalB was an outer membrane protein. To develop an antiserum for protein localization, the ialB gene was cloned in frame into an expression vector with a six-histidine tag and under control of thelacZ promoter. The IalB fusion protein was purified by nickel affinity chromatography and used to raise polyclonal antibodies. IalB was initially localized to the bacterial membrane fraction. To further localize IalB, B. bacilliformis inner and outer membranes were fractionated by sucrose density gradient centrifugation and identified by appearance, buoyant density (ρ), and cytochromeb content. Inner and outer membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and IalB was positively identified by Western blot. Contrary to expectations, IalB was localized to the inner membrane of the pathogen. To directly demonstrate a role for IalB in erythrocyte parasitism, the B. bacilliformis ialB gene was disrupted by insertional mutagenesis. The resulting ialB mutant strain was complemented in trans with a replicative plasmid encoding the full-length ialB gene. PCR and high-stringency DNA hybridization confirmed mutagenesis and transcomplementation events. Abrogation and restoration of ialB expression was verified by SDS-PAGE and immunoblotting. In vitro virulence assays showed that mutagenesis of ialB decreased bacterial association and invasion of human erythrocytes by 47 to 53% relative to controls. Transcomplementation of ialB restored erythrocyte association and invasion rates to levels observed in the parental strain. These data provide direct evidence for IalB's role in erythrocyte parasitism and represent the first demonstration of molecular Koch's postulates for a Bartonella species.

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Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes.

The Journal of Cell Biology ◽

10.1083/jcb.97.5.1582 ◽

1983 ◽

Vol 97 (5) ◽

pp. 1582-1591 ◽

Cited By ~ 71

Author(s):

E S Sztul ◽

K E Howell ◽

G E Palade

Keyword(s):

Membrane Proteins ◽

Golgi Complex ◽

Rat Hepatocytes ◽

Secretory Component ◽

Bile Canaliculus ◽

Sds Page ◽

Iga Antibody ◽

Co Precipitation ◽

Kinetics Of

The intra- and transcellular transports of hepatic secretory and membrane proteins were studied in rats in vivo using [3H]fucose and [35S]cysteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile, and plasma were separated by SDS PAGE and identified by fluorography. 3H-radioactivity in Golgi fractions peaked at 10 min postinjection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from Golgi occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 30 min later than the bulk of content proteins. A major 80,000-dalton form of secretory component (SC) was identified in the bile by co-precipitation with (IgA)2 by an anti-IgA antibody. An antibody (raised in rabbit) against the biliary 80,000-dalton peptide recognized two larger forms (116,000 and 94,000 dalton), presumably precursors, in Golgi membranes. A comparative study of kinetics of transport of 35S-SC and 35S-albumin showed that albumin peaked in bile at approximately 45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins that are delivered to the bile canaliculus.

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