scholarly journals In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane.

1983 ◽  
Vol 96 (3) ◽  
pp. 633-638 ◽  
Author(s):  
D L Paul ◽  
D A Goodenough
Keyword(s):  
Plasma Membranes ◽  
Proteolytic Processing ◽  
Membrane Insertion ◽  
Membrane Association ◽  
Lens Fiber ◽  
In Vitro Synthesis ◽  
Reticulocyte Lysate ◽  
Immune Precipitation ◽  
Dog Pancreas

Synthesis of MP26, the principal protein of lens fiber plasma membranes, was directed in the reticulocyte lysate system by poly A mRNA enriched from whole bovine lens RNA using oligo (dt)-cellulose chromatography. Synthesized MP26 was enriched by immune precipitation. The in vitro-synthesized MP26 had an electrophoretic mobility indistinguishable from that of the native molecule. MP26 showed a cotranslational requirement for dog pancreas microsomes in order for membrane association to occur. Microsome-associated in vitro-synthesized MP26 showed a sensitivity to digestion with chymotrypsin which was similar to the sensitivity of native MP26 in isolated lens fiber plasma membranes, indicating correct insertion of the MP26 into the microsome. Synthesis and membrane insertion of MP26 using N-formyl-[35S]methionyl tRNA as label demonstrated that no proteolytic processing or significant glycosylation accompanied membrane insertion. Chymotryptic cleavage of membrane-inserted, N-formyl-[35S]methionine-labeled MP26 resulted in loss of label, suggesting that the N-terminal of the in vitro-synthesized MP26 faces the cytoplasm.

scholarly journals Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins.

1994 ◽  
Vol 127 (2) ◽  
pp. 343-355 ◽  
Author(s):  
M M Falk ◽  
N M Kumar ◽  
N B Gilula
Keyword(s):  
Plasma Membrane ◽  
Gap Junction ◽  
Proteolytic Processing ◽  
Signal Peptidase ◽  
Membrane Insertion ◽  
Competent Cell ◽  
In Vitro Synthesis ◽  
Amino Terminal

Connexins, the proteins that form gap junction channels, are polytopic plasma membrane (PM) proteins that traverse the plasma membrane bilayer four times. The insertion of five different connexins into the membrane of the ER was studied by synthesizing connexins in translation-competent cell lysates supplemented with pancreatic ER-derived microsomes, and by expressing connexins in vivo in several eucaryotic cell types. In addition, the subcellular distribution of the connexins was determined. In vitro-synthesis in the presence of microsomes resulted in the signal recognition particle-dependent membrane insertion of the connexins. The membrane insertion of all connexins was accompanied by an efficient proteolytic processing that was dependent on the microsome concentration. Endogenous unprocessed connexins were detectable in the microsomes used, indicating that the pancreatic microsomes serve as a competent recipient in vivo for unprocessed full length connexins. Although oriented with their amino terminus in the cytoplasm, the analysis of the cleavage reaction indicated that an unprecedented processing by signal peptidase resulted in the removal of an amino-terminal portion of the connexins. Variable amounts of similar connexin cleavage products were also identified in the ER membranes of connexin overexpressing cells. The amount generated correlated with the level of protein expression. These results demonstrate that the connexins contain a cryptic signal peptidase cleavage site that can be processed by this enzyme in vitro and in vivo in association with their membrane insertion. Consequently, a specific factor or condition must be required to prevent this aberrant processing of connexins under normal conditions in the cell.

Molecular Basis of Salivary Proline-rich Protein and Peptide Synthesis: Cell-free Translations and Processing of Human and Macaque Statherin mRNAs and Partial Amino Acid Sequence of their Signal Peptides

1987 ◽  
Vol 66 (2) ◽  
pp. 462-466 ◽  
Author(s):  
F.G. Oppenheim ◽  
D.I. Hay ◽  
D.J. Smith ◽  
G.D. Offner ◽  
R.F. Troxler
Keyword(s):  
Calcium Phosphate ◽  
Macaca Fascicularis ◽  
Electrophoretic Analysis ◽  
Proteolytic Processing ◽  
Oral Environment ◽  
Reticulocyte Lysate ◽  
Polyprotein Precursor ◽  
Phosphate Salts ◽  
Calcium Phosphate Precipitation

Acidic proline-rich phosphoproteins and phosphopeptides are abundant components of parotid and submandibular salivary secretions in man and in the subhuman primate, Macaca fascicularis. The major acidic proline-rich proteins and the proline-rich phosphopeptide, statherin, of man and macaques have been shown to be potent inhibitors of calcium phosphate precipitation and are thought to function in the oral environment by maintaining saliva supersaturated with respect to calcium phosphate salts. Little is known about the biosynthesis of these proline-rich phosphoproteins and peptides, and the aim of the present work was to determine the structural relationship between statherin precursors and native human and macaque statherin. RNA was isolated from human submandibular gland, and poly(A+) mRNA was selected by affinity chromatography on oligo(dT) cellulose and translated in a reticulocyte lysate. Electrophoretic analysis of the translation products revealed that this mRNA directed the synthesis of a large number of polypeptides with M,s ranging from 5000 to 70,000. Immunoprecipitates, prepared with an antiserum directed against human statherin, contained a single component with a Mr of 7800, approximately 2000 daltons larger than native statherin. Radiosequencing of the in vitro precursor of statherin in immunoprecipitates demonstrated the presence of a 19-residue signal peptide. These results suggest that statherin is derived from a unique structural gene, and does not result from proteolytic processing of a large polyprotein precursor.

Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes

1987 ◽  
Vol 7 (5) ◽  
pp. 1848-1855
Author(s):  
G M Small ◽  
T Imanaka ◽  
H Shio ◽  
P B Lazarow
Keyword(s):  
Candida Tropicalis ◽  
Proteolytic Processing ◽  
In Vitro Translation ◽  
Moderate Number ◽  
Brij 35 ◽  
Reticulocyte Lysate ◽  
Molecular Information ◽  
Acyl Coenzyme A

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.

scholarly journals Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components.

1975 ◽  
Vol 67 (3) ◽  
pp. 852-862 ◽  
Author(s):  
G Blobel ◽  
B Dobberstein
Keyword(s):  
Light Chain ◽  
Proteolytic Processing ◽  
Translation Product ◽  
Globin Mrna ◽  
Ribosomal Subunits ◽  
Initiation Factors ◽  
Detergent Treatment ◽  
Processing Activity ◽  
Dog Pancreas

The data presented in this paper demonstrate that native small ribosomal subunits from reticulocytes (containing initiation factors) and large ribosomal subunits derived from free polysomes of reticulocytes by the puromycin-KCl procedures can function with stripped microsomes derived from dog pancreas rough microsomes in a protein-synthesizing system in vitro in response to added IgG light chain mRNA so as to segregate the translation product in a proteolysis-resistant space. No such segregation took place for the translation product of globin mRNA. In addition to their ability to segregate the translation product of a specific heterologous mRNA, native dog pancreas rough microsomes as well as derived stripped microsomes were able to proteolytically process the larger, primary translation product in an apparently correct manner, as evidenced by the identical mol wt of the segregated translation product and the authentic secreted light chain. Segregation as well as proteolytic processing by native and stripped microsomes occurred only during ongoing translation but not after completion of translation. Attempts to solubilize the proteolytic processing activity, presumably localized in the microsomal membrane by detergent treatment, and to achieve proteolytic processing of the completed light chain precursor protein failed. Taken together, these results establish unequivocally that the information for segregation of a translation product is encoded in the mRNA itself, not in the protein-synthesizing apparatus; this provides strong evidence in support of the signal hypothesis.

In vitro studies on the subcellular location of glucosidase I and glucosidase II in dog pancreas

1986 ◽  
Vol 6 (9) ◽  
pp. 827-834 ◽  
Author(s):  
Ernst Bause ◽  
Roland Günther ◽  
Jürgen Schweden ◽  
Ulrich Tillmann
Keyword(s):  
Molecular Mass ◽  
Consensus Sequence ◽  
Proteolytic Processing ◽  
Translation System ◽  
Translation Product ◽  
Glycosidase Inhibitors ◽  
Glucosidase Ii ◽  
Primary Translation Product ◽  
Dog Pancreas

When programmed with yeast prepro-α-factor mRNA, the heterologous reticulocyte/dog pancreas translation system synthesizes two pheromone related polypeptides, a cytosolically located primary translation product (pp-α-Fcyt, 21 kDa) and a membrane-specific and multiply glycosylated e-factor precursor (pp-α-F3, 27.5 kDa). Glycosylation of the membrane specific pp-α-F3 species is competitively inhibited by synthetic peptides containing the consensus sequence Asn-Xaa-Thr as indicated by a shift of its molecular mass from 27.5 kDa to about 19.5 kDa (pp-α-F0), whereas the primary translation product pp-α-F cyt is not affected. Likewise, only the glycosylated pp-α-F3 structure is digested by Endo H yielding a polypeptide with a molecular mass between PP-α-F0 and pp-α-F cyt. These observations strongly suggest that the primary translation product is proteolytically processed during/on its translocation into the lumen of the microsomal vesicles. We believe that this proteolytic processing is due to the cleavage of a signal sequence from the pp-α-F cyt species, although this interpretation contradicts previous data from other groups. The distinct effect exerted by various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-dNM, 1-deoxymannojirimycin) on the electrophoretic mobility of the pp-α-F3 polypeptide indicates that its oligosaccharide chains are processed to presumbly Man9-GlcNAc2 structures under the in vitro conditions of translation. This oligosaccharide processing is most likely to involve the action of glucosidase I and glucosidase II as follows from the specificity of the glycosidase inhibitors applied and the differences of the molecular mass observed in their presence. In addition, several arguments suggest that both trimming enzymes are located in the lumen of the microsomal vesicles derived from endoplasmic reticulum membranes.

The biogenesis of the cyanellae of Cyanophora paradoxa . III. In vitro synthesis of cyanellar polypeptides using separated cytoplasmic and cyanellar RNA

1989 ◽  
Vol 238 (1290) ◽  
pp. 89-102 ◽  
Keyword(s):  
Protein A ◽  
D1 Protein ◽  
Subcellular Fractionation ◽  
Coupling Factor ◽  
Cyanophora Paradoxa ◽  
In Vitro Synthesis ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate ◽  
Precursor Molecules

RNA from Cyanophora paradoxa was separated into cytoplasmic and cyanellar fractions by using a combination of subcellular fractionation and oligo-dT chromatography. In vitro translation of the separated cyto­plasmic and cyanellar RNAs in a rabbit reticulocyte lysate system in the presence of [ 35 S]methionine resulted in the incorporation of radiolabel into electrophoretically distinct sets of polypeptides. Monospecific and polyspecific antibodies that react with cyanellar polypeptides were used to probe the in vitro translation products by indirect immunoprecipitation by using Staphylococcus protein A conjugated to Sepharose beads. The results indicate that linker polypeptide L1 of the phycobilisome, the γ subunit of coupling factor CF1, and subunit II of PS I are syn­thesized in the cytoplasm as precursor molecules that are 5–8 kDa larger than their mature sizes. Antibodies directed against the psb A gene prod­uct (the D1 protein) precipitated a polypeptide found in the translation products of the cyanellar RNA-directed reactions, which is about 1.5 kDa larger than the mature protein.

A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites

1992 ◽  
Vol 103 (1) ◽  
pp. 131-143 ◽  
Author(s):  
N. Sato ◽  
N. Funayama ◽  
A. Nagafuchi ◽  
S. Yonemura ◽  
S. Tsukita ◽  
...  
Keyword(s):  
Gene Family ◽  
Plasma Membranes ◽  
Immunofluorescence Microscopy ◽  
Membrane Association ◽  
Protein Sequence Analysis ◽  
Teratocarcinoma Cells ◽  
Specific Localization ◽  
End Capping ◽  
Mouse Teratocarcinoma

Radixin is a barbed end-capping actin-modulating protein which was previously reported to be concentrated at cell-to-cell adherens junctions (AJ) and cleavage furrows. Recently, cDNA encoding mouse radixin was isolated, showing that radixin is highly homologous to but distinct from ezrin. From mouse teratocarcinoma cells we isolated and analyzed cDNA encoding another radixin-related protein. Sequence analysis has demonstrated that this protein is a mouse homologue of human moesin (98.3% identity) and that it shares 71.7% and 80.1% identity with ezrin and radixin, respectively. Translation experiments in vitro combined with immunoblot analyses led us to conclude that there is a gene family consisting of ezrin, radixin and moesin. These members are coexpressed in various types of cells. Then, by immunofluorescence microscopy, we closely analyzed their distribution using polyclonal and monoclonal antibodies, which could recognize all three members. In addition to cell-to-cell AJ and cleavage furrows, it was shown that they were concentrated at microvilli and ruffling membranes in various types of cells. Furthermore, the cell-to-substrate AJ (focal contacts) were clearly stained by anti-radixin pAb only after the apical/lateral membranes and cytoplasm were removed by the zinc method. We conclude that at least one of the members of the ezrin-radixin-moesin family is concentrated at specific regions where actin filaments are densely associated with plasma membranes.

scholarly journals Efficient association of in vitro translation products with purified stable Candida tropicalis peroxisomes.

1987 ◽  
Vol 7 (5) ◽  
pp. 1848-1855 ◽  
Author(s):  
G M Small ◽  
T Imanaka ◽  
H Shio ◽  
P B Lazarow
Keyword(s):  
Candida Tropicalis ◽  
Proteolytic Processing ◽  
In Vitro Translation ◽  
Moderate Number ◽  
Brij 35 ◽  
Reticulocyte Lysate ◽  
Molecular Information ◽  
Acyl Coenzyme A

Newly synthesized peroxisomal proteins enter preexisting peroxisomes posttranslationally in vivo, generally without proteolytic processing. An efficient reconstitution of this process in vitro together with cloned DNAs for peroxisomal proteins would make possible investigation of the molecular information that targets proteins to peroxisomes. We have previously reported the isolation of clones for Candida tropicalis peroxisomal proteins; here we describe the association (and possible import) of peroxisomal proteins with peroxisomes in vitro. C. tropicalis was grown in a medium containing Brij 35, resulting in the induction of a moderate number of medium-sized peroxisomes. These peroxisomes, isolated in a sucrose gradient, had a catalase latency of 54% and were sufficiently stable to be concentrated and used in an import assay. The reticulocyte lysate translation products of total RNA from oleate-grown cells were incubated with the peroxisomes at 26 degrees C in the presence of 50 mM KCl, protease inhibitors, 0.5 M sucrose, 2.5 mM MOPS (morpholinepropanesulfonic acid) (pH 7.2), and 0.5 mM EDTA. Ten major translation products (which could be immunoprecipitated with antiserum against peroxisomal protein) became progressively associated with the peroxisomes during the first 30 min of incubation (some up to approximately 70%). These include acyl coenzyme A oxidase and the trifunctional protein hydratase-dehydrogenase-epimerase. This association did not occur at 4 degrees C nor did it occur if the peroxisomes were replaced with mitochondria.

scholarly journals Junctions between lens fiber cells are labeled with a monoclonal antibody shown to be specific for MP26.

1985 ◽  
Vol 100 (1) ◽  
pp. 216-225 ◽  
Author(s):  
D F Sas ◽  
M J Sas ◽  
K R Johnson ◽  
A S Menko ◽  
R G Johnson
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membranes ◽  
Antigenic Site ◽  
Ultrastructural Level ◽  
Protein Band ◽  
Cell Contact ◽  
Lens Fiber ◽  
Lens Fiber Cell ◽  
Transfer Procedures

A monoclonal antibody (mcAb) that recognizes an intracellular domain of the major lens membrane protein in both chicken and bovine lenses is described. Mice were immunized with chicken lens fiber cell membranes that had been washed with 7 M urea. Hybridomas were screened by means of enzyme-linked immunosorbent assays and the molecular specificities of the mcAbs were determined using electrophoretic transfer procedures, "Westerns." One of these mcAbs, an IgG designated B2, reacted with a single band of 28,000 Mr from the chicken embryo lens (MP28) and the analogous 26,000 Mr protein in the bovine lens (MP26). Monoclonal B2 was shown to be specific for these proteins, since (a) heating in SDS caused MP26 to aggregate and reduced B2 binding to the protein band at an Mr of 26,000 in Western transfer analysis; (b) apparent dimers were bound by B2 in Western transfers; (c) soluble protein fractions from the lens contained no detectable B2 antigens; and (d) a cyanogen bromide fragment of MP26 was bound by B2. Studies with several proteases indicated that the antigenic site for B2 resides on a 2-kd, protease-sensitive region at the C-terminal end of MP26 and MP28. Evidence for B2 binding on the cytoplasmic side of the membrane comes from labeling studies done at the ultrastructural level. These studies, utilizing indirect methods with peroxidase and colloidal gold markers, clearly demonstrated that B2 labels two types of junctional profiles. In our calf lens membrane preparations after tannic acid staining, the predominant type (80%) measured 16-18 nn thick, with the second type measuring only 12-14 nm. Chick embryo lens cells that had differentiated in vitro and formed groups of lens fiber-like cells (termed lentoids), fluoresced brightly only when they had been permeabilized before labeling with B2 and a fluorochrome-conjugated antibody. This binding was concentrated at the plasma membranes of cells within the lentoids, even outside areas of cell-cell contact. Surrounding epithelioid cells were not stained. Solubilized lens cultures, examined by Westerns, displayed a single immunoreactive band, which co-migrated with MP28.

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