scholarly journals Early disulfide bond formation prevents heterotypic aggregation of membrane proteins in a cell-free translation system.

1992 ◽  
Vol 118 (2) ◽  
pp. 245-252 ◽  
Author(s):  
M Yilla ◽  
D Doyle ◽  
J T Sawyer
Keyword(s):  
Membrane Proteins ◽  
Transmembrane Domain ◽  
Binding Activity ◽  
Translation System ◽  
Folding Intermediates ◽  
Free Translation ◽  
Heterogeneous Interaction ◽  
A Cell ◽  
Reduced State

We previously demonstrated that a heterotypic complex of the two rat asialoglycoprotein receptor subunits was assembled during cell-free translation (Sawyer, J. T., and D. Doyle. 1990. Proc. Natl. Acad. Sci. USA. 87:4854-4858). We have characterized this system further by analyzing polypeptide interactions under both reducing and oxidizing translation conditions. This report shows that the complex represents a heterogeneous interaction between reduced membrane proteins rather than a specific oligomeric structure. In the reduced state membrane proteins interact in this system to form aggregates of diverse size and composition. The aggregated nascent polypeptides interact with the immunoglobulin heavy chain binding protein but this protein is not an integral component of the aggregate. Aggregation occurs via the exoplasmic domain, rather than the transmembrane domain, and the folding of this domain by the formation of intramolecular disulfides, prevents the interaction from occurring. Additionally, the folded molecules containing intramolecular disulfides lack high affinity binding activity and thus appear to resemble the earliest folding intermediates seen in vivo (Olson, J. T., and M. D. Lane. 198. FASEB (Fed. Am. Soc. Exp. Biol.) J. 3:1618-1624). These results lead us to suggest that the formation of intramolecular disulfides during early biogenesis serves to prevent nonspecific associations between nascent polypeptides.

Unusually Severe Heterozygous β-Thalassemia: Evidence for an Interacting Gene Affecting Globin Translation

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3428-3435 ◽  
Author(s):  
P. Joy Ho ◽  
Georgina W. Hall ◽  
Suzanne Watt ◽  
Nicholas C. West ◽  
Jennifer W. Wimperis ◽  
...  
Keyword(s):  
Dominant Mode ◽  
Translation System ◽  
Severe Phenotype ◽  
Asian Populations ◽  
Free Translation ◽  
A Cell ◽  
Chain Imbalance ◽  
American Society ◽  
Intron 2

A common β-thalassemia mutation in Asian populations is the C → T substitution at position 654 of intron 2, which leads to the activation of two cryptic splicing sites and the incorporation of 73 extra nucleotides into the mutant mRNA. Like most β-thalassemia mutations, it normally exhibits recessive inheritance. We investigated the unusually severe phenotype in two heterozygotes for this mutation, father and son, who had thalassemia intermedia and an apparent dominant mode of inheritance. An increased level of aberrantly spliced transcript in the reticulocytes of the probands compared with asymptomatic β654heterozygotes led us to investigate the production and processing of β654 RNA. We showed that large amounts of the aberrant β654 transcript were detectable in erythroblasts from one of the asymptomatic cases. The translation product of this mRNA was not detectable in vivo, and we were unable to demonstrate the translation of the mutant mRNA in a cell-free translation system. Although the reticulocyte :β mRNA ratios in the two probands were within the range observed in the asymptomatic heterozygotes, globin chain biosynthesis studies showed that the probands had considerably greater :β chain imbalance. These results imply that the more severe phenotype may be due to a second defect, possibly unlinked to the β-globin cluster, that acts at the translational or posttranslational level. © 1998 by The American Society of Hematology.

Unusually Severe Heterozygous β-Thalassemia: Evidence for an Interacting Gene Affecting Globin Translation

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3428-3435 ◽  
Author(s):  
P. Joy Ho ◽  
Georgina W. Hall ◽  
Suzanne Watt ◽  
Nicholas C. West ◽  
Jennifer W. Wimperis ◽  
...  
Keyword(s):  
Dominant Mode ◽  
Translation System ◽  
Severe Phenotype ◽  
Asian Populations ◽  
Free Translation ◽  
A Cell ◽  
Chain Imbalance ◽  
American Society ◽  
Intron 2

Abstract A common β-thalassemia mutation in Asian populations is the C → T substitution at position 654 of intron 2, which leads to the activation of two cryptic splicing sites and the incorporation of 73 extra nucleotides into the mutant mRNA. Like most β-thalassemia mutations, it normally exhibits recessive inheritance. We investigated the unusually severe phenotype in two heterozygotes for this mutation, father and son, who had thalassemia intermedia and an apparent dominant mode of inheritance. An increased level of aberrantly spliced transcript in the reticulocytes of the probands compared with asymptomatic β654heterozygotes led us to investigate the production and processing of β654 RNA. We showed that large amounts of the aberrant β654 transcript were detectable in erythroblasts from one of the asymptomatic cases. The translation product of this mRNA was not detectable in vivo, and we were unable to demonstrate the translation of the mutant mRNA in a cell-free translation system. Although the reticulocyte :β mRNA ratios in the two probands were within the range observed in the asymptomatic heterozygotes, globin chain biosynthesis studies showed that the probands had considerably greater :β chain imbalance. These results imply that the more severe phenotype may be due to a second defect, possibly unlinked to the β-globin cluster, that acts at the translational or posttranslational level. © 1998 by The American Society of Hematology.

scholarly journals Assembly associated with the cytomatrix.

1984 ◽  
Vol 99 (1) ◽  
pp. 209s-211s ◽  
Author(s):  
A B Fulton
Keyword(s):  
Giant Cells ◽  
Cytoskeletal Proteins ◽  
Translation System ◽  
Free Translation ◽  
A Cell

Assembly in vivo has been studied both for endogenous cytoskeletal proteins and for several classes of viruses. Autoradiography of cytoskeletal proteins has shown that many associate with the cytoskeletal framework close to the time and place of synthesis. The cytoskeletal proteins rearrange after association with the cytoskeletal framework. Rearrangement in symmetrical giant cells occurs in a centrifugal and coherent pattern. Many of the cytoskeletal proteins associate cotranslationally, as shown by their puromycin resistance in a cell-free translation system. The assembly of several groups of viruses has been shown to be associated with various components of the cytoskeleton; whether such assembly is cotranslational has not yet been addressed directly.

scholarly journals The 3' untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro.

1993 ◽  
Vol 13 (6) ◽  
pp. 3340-3349 ◽  
Author(s):  
X Danthinne ◽  
J Seurinck ◽  
F Meulewaeter ◽  
M Van Montagu ◽  
M Cornelissen
Keyword(s):  
Tobacco Necrosis Virus ◽  
Translation System ◽  
Coding Region ◽  
Necrosis Virus ◽  
Untranslated Sequence ◽  
Virus Rna ◽  
Free Translation ◽  
Order Of Magnitude ◽  
A Cell

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.

scholarly journals A murine fer testis-specific transcript (ferT) encodes a truncated Fer protein.

1990 ◽  
Vol 10 (1) ◽  
pp. 146-153 ◽  
Author(s):  
K Fischman ◽  
J C Edman ◽  
G M Shackleford ◽  
J A Turner ◽  
W J Rutter ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Translation System ◽  
Appearance Time ◽  
Acid Protein ◽  
Mouse Tissues ◽  
Free Translation ◽  
Alternatively Spliced ◽  
A Cell ◽  
Testis Cdna ◽  
Carboxy Terminal

A cDNA for a potential tyrosine kinase-encoding mRNA was isolated from a mouse testis cDNA library. In a survey of eight mouse tissues, a transcript of 2.4 kilobases restricted to testis tissue was found. The mRNA encodes a 453-amino-acid protein of 51,383 daltons, the smallest tyrosine kinase protein ever described. RNA synthesized from the cDNA template directs the synthesis of a 51,000-Mr protein in a cell-free translation system. The carboxy-terminal 409 amino acids are 98 and 90% identical to the carboxy halves of the rat and human Fer proteins, respectively. This suggests that the cDNA represents an alternatively spliced testis-specific fer mRNA and is therefore termed by us ferT. On the basis of the appearance time of the fer mRNA in the testis of maturing neonatal mice, we speculate on the role played by this protein in the development of this organ.

scholarly journals Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system

2011 ◽  
Vol 11 (1) ◽  
pp. 35 ◽  
Author(s):  
Akira Nozawa ◽  
Tomio Ogasawara ◽  
Satoko Matsunaga ◽  
Takahiro Iwasaki ◽  
Tatsuya Sawasaki ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Partial Purification ◽  
Translation System ◽  
Free Translation
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