scholarly journals Posttranslational incorporation of contractile proteins into myofibrils in a cell-free system.

1988 ◽  
Vol 107 (2) ◽  
pp. 587-596 ◽  
Author(s):  
M Bouché ◽  
S M Goldfine ◽  
D A Fischman
Keyword(s):  
In Vitro Translation ◽  
Homologous Proteins ◽  
Free System ◽  
Cell Free System ◽  
Reticulocyte Lysate ◽  
Thin Filament Proteins ◽  
A Cell ◽  
The Many ◽  
Kinetic Equilibrium

The incorporation of newly synthesized protein into myofibrils has been examined in a cell-free system. Myofibrils were added to a reticulocyte lysate after the in vitro translation of muscle-specific poly(A)+RNA. Only a small number of the many synthesized proteins were found to associate with the exogenously added myofibrils. These proteins were all identified as sarcomeric components and had subunit mobilities (Mr) of 200, 140, 95, 86, 43, 38, 35, 25, 23, 20, and 18 kD. The association was rapid (t1/2 less than 15 min) and, for most of the proteins, relatively temperature insensitive. Except for a 43-kD polypeptide, tentatively identified as beta-actin, none of the proteins encoded by brain poly(A)+RNA associated with the myofibrils. When filaments made from purified myosin or actin were used as the "capture" substrates, only thick or thin filament proteins, respectively, were incorporated. Incorporation was substantially reduced when cross-linked myosin filaments were used. These results are compatible with a model in which proteins of the sarcomere are in kinetic equilibrium with homologous proteins in a soluble pool.

scholarly journals SMIM1, carrier of the Vel blood group, is a tail-anchored transmembrane protein and readily forms homodimers in a cell-free system

2020 ◽  
Vol 40 (5) ◽  
Author(s):  
Anja Nylander ◽  
Pawel Leznicki ◽  
Karina Vidovic ◽  
Stephen High ◽  
Martin L. Olsson
Keyword(s):  
Blood Group ◽  
Transmembrane Protein ◽  
Membrane Orientation ◽  
Free System ◽  
Cell Free System ◽  
C Terminus ◽  
Reticulocyte Lysate ◽  
A Cell ◽  
Future Work

Abstract Antibodies to the Vel blood group antigen can cause adverse hemolytic reactions unless Vel-negative blood units are transfused. Since the genetic background of Vel-negativity was discovered in 2013, DNA-based typing of the 17-bp deletion causing the phenotype has facilitated identification of Vel-negative blood donors. SMIM1, the gene underlying Vel, encodes a 78-amino acid erythroid transmembrane protein of unknown function. The transmembrane orientation of SMIM1 has been debated since experimental data supported both the N- and C-termini being extracellular. Likewise, computational predictions of its orientation were divided and potential alternatives such as monotopic or dual-topology have been discussed but not investigated. We used a cell-free system to explore the topology of SMIM1 when synthesized in the endoplasmic reticulum (ER). SMIM1 was tagged with an opsin-derived N-glycosylation reporter at either the N- or C-terminus and synthesized in vitro using rabbit reticulocyte lysate supplemented with canine pancreatic microsomes as a source of ER membrane. SMIM1 topology was then determined by assessing the N-glycosylation of its N- or C-terminal tags. Complementary experiments were carried out by expressing the same SMIM1 variants in HEK293T/17 cells and establishing their membrane orientation by immunoblotting and flow cytometry. Our data consistently indicate that SMIM1 has its short C-terminus located extracellularly and that it most likely belongs to the tail-anchored class of membrane proteins with the bulk of the polypeptide located in the cytoplasm. Having established its membrane orientation in an independent model system, future work can now focus on functional aspects of SMIM1 as a potential regulator of erythropoiesis.

Biotin in vitro Translation: A Nonradioactive Method for the Synthesis of Biotin Labeled Proteins in a Cell-Free System

1996 ◽  
pp. 183-199
Author(s):  
Hans-Joachim Hoeltke ◽  
Irene Ettl ◽  
Edith Strobel ◽  
Hermann Leying ◽  
Maria Zimmermann ◽  
...  
Keyword(s):  
In Vitro Translation ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Reconstitution of the folding pathway of collagen in a cell-free system: formation of correctly aligned and hydroxylated triple helices

1993 ◽  
Vol 296 (2) ◽  
pp. 511-517 ◽  
Author(s):  
R B Middleton ◽  
N J Bulleid
Keyword(s):  
Post Translational Modification ◽  
Type X Collagen ◽  
Free System ◽  
Cell Free System ◽  
Native Collagen ◽  
Reticulocyte Lysate ◽  
A Cell ◽  
Band Characteristic

We describe here a cell-free system which will carry out the initial stages in the synthesis, post-translational modification and assembly of type-X collagen. The mRNA coding for bovine type-X collagen was synthesized in vitro and translated in a rabbit reticulocyte lysate to yield a protein that was collagenase sensitive and could be immunoprecipitated with antibodies raised to purified avian type-X collagen. When type-X collagen was synthesized in the absence of added microsomes or in the presence of canine pancreas microsomes, the translation products showed partial resistance to digestion with pepsin but were completely degraded with a mixture of chymotrypsin and trypsin, suggesting that only incorrectly aligned non-native collagen molecules were synthesized under these conditions. When the protein was synthesized in the presence of microsomes derived from avian fibroblasts or a human fibrosarcoma cell line, the translocated product migrated as a diffuse band characteristic of hydroxylated collagen. The synthesized polypeptides were also resistant to both pepsin and trypsin/chymotrypsin digestion, demonstrating the formation of correctly aligned native collagen. Furthermore, the collagen polypeptides assembled into higher-order structures, possibly trimers, which were stabilized by interchain disulphide bonds. The collagen helix synthesized in vitro had a melting temperature of 41 degrees C which is comparable with the protein synthesized in vivo, further demonstrating that the polypeptides were hydroxylated and that the triple helix formed was correctly aligned.

A somatic cell-derived system for studying both early and late mitotic events in vitro

1989 ◽  
Vol 94 (3) ◽  
pp. 449-462
Author(s):  
J. Nakagawa ◽  
G.T. Kitten ◽  
E.A. Nigg
Keyword(s):  
Chromatin Condensation ◽  
Nuclear Lamina ◽  
Free System ◽  
Cell Free System ◽  
Protein Substrates ◽  
Nuclear Envelope Breakdown ◽  
Biochemical Fractionation ◽  
A Cell

We describe a cell-free system for studying mitotic reorganization of nuclear structure. The system utilizes soluble extracts prepared from metaphase-arrested somatic chicken cells and supports both the disassembly and subsequent partial reassembly of exogenous nuclei. By fluorescence microscopy, biochemical fractionation, protein phosphorylation assays and electron microscopy, we show that chicken embryonic nuclei incubated in extracts prepared from metaphase-arrested chicken hepatoma cells undergo nuclear envelope breakdown, lamina depolymerization and chromatin condensation. These prophase-like events are strictly dependent on ATP and do not occur when nuclei are incubated in interphase extracts. Compared to interphase extracts, metaphase extracts show increased kinase activities toward a number of nuclear protein substrates, including lamins and histone H1; moreover, they specifically contain four soluble phosphoproteins of Mr 38,000, 75,000, 95,000 and 165,000. Following disassembly of exogenous nuclei in metaphase extracts, telophase-like reassembly of a nuclear lamina and re-formation of nuclear membranes around condensed chromatin can be induced by depletion of ATP from the extract. We anticipate that this reversible cell-free system will contribute to the identification and characterization of factors involved in regulatory and mechanistic aspects of mitosis.

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