scholarly journals Translational control of insulin biosynthesis. Evidence for regulation of elongation, initiation and signal-recognition-particle-mediated translational arrest by glucose

1986 ◽  
Vol 235 (2) ◽  
pp. 459-467 ◽  
Author(s):  
M Welsh ◽  
N Scherberg ◽  
R Gilmore ◽  
D F Steiner
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Blot Hybridization ◽  
Signal Recognition Particle ◽  
Chain Elongation ◽  
Insulin Biosynthesis ◽  
Signal Recognition ◽  
Insulin Synthesis ◽  
Run Off ◽  
Insulin Mrna

The biosynthesis of insulin in the islets of Langerhans is strongly controlled at the translational level by glucose. We have used a variety of experimental approaches in efforts to dissect the mechanisms underlying the stimulatory effect of glucose. To assess its effects on rates of peptide-chain elongation, isolated rat islets were labelled with [3H]leucine at different glucose concentrations in the presence or absence of low concentrations of cycloheximide. Under these conditions, at glucose concentrations up to 5.6 mM, endogenous insulin mRNA did not become rate-limiting for the synthesis of insulin, whereas stimulation of non-insulin protein synthesis was abolished by cycloheximide at all glucose concentrations, indicating either that insulin synthesis is selectively regulated at the level of elongation at glucose concentrations up to 5.6 mM, or that at these concentrations inactive insulin mRNA is transferred to an actively translating pool. Glucose-induced changes in the intracellular distribution of insulin mRNA in cultured islets were assessed by subcellular fractionation and blot-hybridization using insulin cDNA probes. At glucose concentrations above 3.3 mM, cytoplasmic insulin mRNA was increasingly transferred to fractions co-sedimenting with ribosomes, and relatively more of the ribosome-associated insulin mRNA became membrane-associated, consistent with effects of glucose above 3.3 mM on both the initiation of insulin mRNA and SRP (signal recognition particle)-mediated transfer of cytosolic nascent preproinsulin to the endoplasmic reticulum. When freshly isolated islets were homogenized and incubated with 125I-Tyr-tRNA, run-off incorporation of 125I into preproinsulin was increased by prior incubation of the islets at 16.7 mM-glucose. The addition of purified SRP receptor increased the run-off incorporation of [125I]iodotyrosine into preproinsulin, especially when the islets had been preincubated at 16.7 mM-glucose. These findings taken together suggest that glucose may stimulate elongation rates of nascent preproinsulin at concentrations up to 5.6 mM, stimulates initiation of protein synthesis involving both insulin and non-insulin mRNA at concentrations above 3.3 mM, and increases the transfer of initiated insulin mRNA molecules from the cytoplasm to microsomal membranes by an SRP-mediated mechanism that involves the modification of interactions between SRP and its receptor.

scholarly journals GTP-binding proteins may stimulate insulin biosynthesis in rat pancreatic islets by enhancing the signal-recognition-particle-dependent translocation of the insulin mRNA poly-/mono-some complex to the endoplasmic reticulum

1991 ◽  
Vol 275 (1) ◽  
pp. 23-28 ◽  
Author(s):  
N Welsh ◽  
C Oberg ◽  
M Welsh
Keyword(s):  
Endoplasmic Reticulum ◽  
Binding Proteins ◽  
Specific Binding ◽  
Signal Recognition Particle ◽  
Insulin Biosynthesis ◽  
Signal Recognition ◽  
Insulin Synthesis ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Insulin Mrna

We aimed to elucidate the putative role of GTP-binding proteins in the regulation of insulin biosynthesis. For this purpose, freshly isolated rat islets were incubated in the presence of liposomes containing GDP, guanosine 5′-[beta-thio]diphosphate (GDP[S]), GTP, guanosine 5′-[gamma-thio]triphosphate (GTP[S]), guanosine 5′-[beta gamma-methylene]triphosphate (p[CH2]ppG), guanosine 5′[beta gamma-imido]triphosphate (p[NH]ppG) and ATP, and the effects of the liposomal delivery of these substances on rates of biosynthesis of insulin and total protein were determined. Insulin biosynthesis during a 1 h incubation at 1.67 mM-glucose was stimulated by ATP- and GTP[S]-containing liposomes as compared with control liposomes. At 16.7 mM-glucose, only the GTP[S]-containing liposomes stimulated insulin biosynthesis. No inhibition of islet protein and insulin synthesis was observed with GDP-, GDP[S]-, p[CH2]ppG- and p[NH]ppG-containing liposomes. By determining the subcellular distribution of insulin mRNA, it was found that the mRNA content associated with microsomes was increased and that associated with the cytosolic mono-/poly-somes decreased when the islets were incubated with GTP[S]-containing liposomes, resulting in an approximate doubling of the ratio of microsomal to polysomal-associated insulin mRNA. ATP-containing liposomes produced no effects on the association of insulin mRNA with microsomes. By using photoaffinity labelling and immunoprecipitation techniques, specific binding of GTP[35S] to the alpha-subunit of the signal-recognition particle (SRP) receptor in islet homogenates containing physiological concentrations of GTP and GDP was demonstrated. These findings suggest that the GTP-binding subunit(s) of the SRP receptor, and possibly also of other GTP-binding proteins involved in this process, may regulate insulin biosynthesis by stimulating the translocation of insulin mRNA to the endoplasmic reticulum and by increasing preproinsulin-peptide translocation into the lumen of the reticulum.

scholarly journals Signal recognition particle causes a transient arrest in the biosynthesis of prepromelittin and mediates its translocation across mammalian endoplasmic reticulum.

1987 ◽  
Vol 104 (1) ◽  
pp. 61-66 ◽  
Author(s):  
I Ibrahimi
Keyword(s):  
Endoplasmic Reticulum ◽  
Wheat Germ ◽  
Signal Recognition Particle ◽  
Chain Elongation ◽  
Translation System ◽  
Signal Recognition ◽  
Nascent Chain ◽  
Reticulocyte Lysate ◽  
The Difference ◽  
Chain Synthesis

The translocation of prepromelittin (pPM) across mammalian endoplasmic reticulum was studied in both wheat germ and reticulocyte lysate. In the wheat germ system, signal recognition particle (SRP) caused a transient arrest in the synthesis of pPM. This was indicated by a slowdown in the rate of synthesis of pPM in the presence of SRP. The arrest was specific, dependent on the concentration of SRP, and more effective at early incubation time. In a tightly synchronized translation system, SRP had no apparent effect on the elongation of pPM, indicating that the effect of SRP on pPM chain synthesis might be at the final stages of chain elongation and release from the ribosome. This was reflected in a transient accumulation of pPM as peptidyl tRNA. Because pPM is composed of only 70 amino acids, arrest by SRP may be very close to chain termination. Arrest at this stage of chain synthesis seems to be unstable and the nascent chain gets terminated and released from the ribosome after a transient delay. The translocation of pPM was shown to be dependent on both SRP and docking protein. The difference in the translocation efficiency of pPM in reticulocyte and wheat germ lysates may reflect a difference in the targeting process in the two systems.

scholarly journals The SRP9/14 subunit of the signal recognition particle (SRP) is present in more than 20-fold excess over SRP in primate cells and exists primarily free but also in complex with small cytoplasmic Alu RNAs.

1995 ◽  
Vol 6 (4) ◽  
pp. 471-484 ◽  
Author(s):  
F Bovia ◽  
M Fornallaz ◽  
H Leffers ◽  
K Strub
Keyword(s):  
Translational Control ◽  
Small Rnas ◽  
Control Function ◽  
Signal Recognition Particle ◽  
Synthesis Rate ◽  
Signal Recognition ◽  
Control Of Gene Expression ◽  
Fold Excess ◽  
Srp Rna

The heterodimeric protein SRP9/14 bound to the Alu sequences of SRP RNA is essential for the translational control function of the signal recognition particle (SRP). The Alu RNAs of primate cells are believed to be derived from SRP RNA and have been shown to bind to an SRP14-related protein in vitro. We have used antibodies to characterize SRP9/14 and examine its association with small RNAs in vivo. Although SRP9 proteins are the same size in both rodent and primate cells, SRP14 subunits are generally larger in primate cells. An additional alanine-rich domain at the C-terminus accounts for the larger size of one human isoform. Although the other four SRP proteins are largely assembled into SRP in both rodent and primate cells, we found that the heterodimer SRP9/14 is present in 20-fold excess over SRP in primate cells. An increased synthesis rate of both proteins may contribute to their accumulation. The majority of the excess SRP9/14 is cytoplasmic and does not appear to be bound to any small RNAs; however, a significant fraction of a small cytoplasmic Alu RNA is complexed with SRP9/14 in a 8.5 S particle. Our findings that there is a large excess of SRP9/14 in primate cells and that Alu RNAs are bound to SRP9/14 in vivo suggest that this heterodimeric protein may play additional roles in the translational control of gene expression and/or Alu transcript metabolism.

Pathways for compartmentalizing protein synthesis in eukaryotic cells: the template-partitioning model

2005 ◽  
Vol 83 (6) ◽  
pp. 687-695 ◽  
Author(s):  
Christopher V Nicchitta ◽  
Rachel S Lerner ◽  
Samuel B Stephens ◽  
Rebecca D Dodd ◽  
Brook Pyhtila
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Eukaryotic Cell ◽  
Signal Recognition ◽  
Cytosolic Proteins ◽  
Complex Process ◽  
Hypothetical Mechanism ◽  
Cytosolic Ribosomes

mRNAs encoding signal sequences are translated on endoplasmic reticulum (ER) - bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on cytosolic ribosomes. The partitioning of mRNAs to the ER occurs by positive selection; cytosolic ribosomes engaged in the translation of signal-sequence-bearing proteins are engaged by the signal-recognition particle (SRP) pathway and subsequently trafficked to the ER. Studies have demonstrated that, in addition to the SRP pathway, mRNAs encoding cytosolic proteins can also be partitioned to the ER, suggesting that RNA partitioning in the eukaryotic cell is a complex process requiring the activity of multiple RNA-partitioning pathways. In this review, key findings on this topic are discussed, and the template-partitioning model, describing a hypothetical mechanism for RNA partitioning in the eukaryotic cell, is proposed.Key words: mRNA, ribosome, endoplasmic reticulum, translation, protein synthesis, signal sequence, RNA localization.

scholarly journals Full-length prepro-alpha-factor can be translocated across the mammalian microsomal membrane only if translation has not terminated.

1988 ◽  
Vol 106 (4) ◽  
pp. 1043-1048 ◽  
Author(s):  
P D Garcia ◽  
P Walter
Keyword(s):  
Protein Synthesis ◽  
Signal Recognition Particle ◽  
Full Length ◽  
Microsomal Membrane ◽  
Signal Recognition ◽  
Translational Machinery ◽  
Alpha Factor ◽  
Polypeptide Chains

We have previously shown that fully synthesized prepro-alpha-factor (pp alpha F), the precursor for the yeast pheromone alpha-factor, can be translocated posttranslationally across yeast rough microsomal (RM) membranes from a soluble, ribosome-free pool. We show here that this is not the case for translocation of pp alpha F across mammalian RM. Rather we found that a small amount of translocation of full-length pp alpha F is observed, but is solely due to polypeptide chains that were still ribosome bound and covalently attached to tRNA, i.e., not terminated. In addition, both signal recognition particle (SRP) and SRP receptor are required, i.e., the same targeting machinery that is normally responsible for the coupling between protein synthesis and translocation. Thus, the molecular requirements for targeting are distinct from posttranslational translocation across yeast RM. As termination is generally regarded as part of translation, the translocation of full-length pp alpha F across mammalian RM does not occur "posttranslationally," albeit independent of elongation. Most other proteins for which posttranslational translocation across mammalian RM was previously claimed fall into the same category in that ribosome attachment as peptidyl-tRNA is required. To clearly separate these two distinct processes, we suggest that the term posttranslational be reserved for those processes that occur in the complete absence of the translational machinery. We propose the term "ribosome-coupled translocation" for the events described here.

scholarly journals Depletion of the Signal Recognition Particle Receptor Inactivates Ribosomes in Escherichia coli

2009 ◽  
Vol 191 (22) ◽  
pp. 7017-7026 ◽  
Author(s):  
Jonas Bürk ◽  
Benjamin Weiche ◽  
Meike Wenk ◽  
Diana Boy ◽  
Sigrun Nestel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Ribosome Biogenesis ◽  
Protein Quality ◽  
Signal Recognition Particle ◽  
Eukaryotic Cells ◽  
Striking Difference ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
E Coli

ABSTRACT The signal recognition particle (SRP)-dependent cotranslational targeting of proteins to the cytoplasmic membrane in bacteria or the endoplasmic reticulum membrane in eukaryotes is an essential process in most living organisms. Eukaryotic cells have been shown to respond to an impairment of the SRP pathway by (i) repressing ribosome biogenesis, resulting in decreased protein synthesis, and (ii) by increasing the expression of protein quality control mechanisms, such as chaperones and proteases. In the current study, we have analyzed how bacteria like Escherichia coli respond to a gradual depletion of FtsY, the bacterial SRP receptor. Our analyses using cell-free transcription/translation systems showed that FtsY depletion inhibits the translation of both SRP-dependent and SRP-independent proteins. This synthesis defect is the result of a multifaceted response that includes the upregulation of the ribosome-inactivating protein ribosome modulation factor (RMF). Although the consequences of these responses in E. coli are very similar to some of the effects also observed in eukaryotic cells, one striking difference is that E. coli obviously does not reduce the rate of protein synthesis by downregulating ribosome biogenesis. Instead, the upregulation of RMF leads to a direct and reversible inhibition of translation.

scholarly journals Multifaceted Physiological Response Allows Yeast to Adapt to the Loss of the Signal Recognition Particle-dependent Protein-targeting Pathway

2001 ◽  
Vol 12 (3) ◽  
pp. 577-588 ◽  
Author(s):  
Sarah C. Mutka ◽  
Peter Walter
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Heat Shock ◽  
Cell Growth ◽  
Physiological Response ◽  
Protein Translocation ◽  
Protein Sorting ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Dependent Protein

Translational control has recently been recognized as an important facet of adaptive responses to various stress conditions. We describe the adaptation response of the yeast Saccharomyces cerevisiae to the loss of one of two mechanisms to target proteins to the secretory pathway. Using inducible mutants that block the signal recognition particle (SRP) pathway, we find that cells demonstrate a physiological response to the loss of the SRP pathway that includes specific changes in global gene expression. Upon inducing the loss of the SRP pathway, SRP-dependent protein translocation is initially blocked, and cell growth is considerably slowed. Concomitantly, gene expression changes include the induction of heat shock genes and the repression of protein synthesis genes. Remarkably, within hours, the efficiency of protein sorting improves while cell growth remains slow in agreement with the persistent repression of protein synthesis genes. Our results suggest that heat shock gene induction serves to protect cells from mislocalized precursor proteins in the cytosol, whereas reduced protein synthesis helps to regain efficiency in protein sorting by reducing the load on the protein translocation apparatus. Thus, we suggest that cells trade speed in cell growth for fidelity in protein sorting to adjust to life without SRP.

scholarly journals Effects of a diabetogenic strain of encephalomyocarditis (EMC) virus on protein synthesis in mouse islets of Langerhans

1990 ◽  
Vol 270 (3) ◽  
pp. 777-781 ◽  
Author(s):  
T Ward ◽  
M J Clemens ◽  
K W Taylor
Keyword(s):  
Protein Synthesis ◽  
Insulin Release ◽  
Post Infection ◽  
Protein Biosynthesis ◽  
Insulin Biosynthesis ◽  
Mrna Levels ◽  
Gapdh Mrna ◽  
Emc Virus ◽  
Insulin Mrna ◽  
Mouse Islets

The effects of a diabetogenic strain of encephalomyocarditis (EMC) virus on total protein and insulin biosynthesis in mouse islets of Langerhans have been studied in tissue culture. In dispersed mouse islets, the rates of protein biosynthesis were assessed by measuring the incorporation of [3H]leucine into proteins. In infected dispersed islets incubated in 20 mM-glucose, both insulin and total protein biosynthesis were decreased at 6 h; only insulin biosynthesis was significantly decreased at 3 h. In whole islets, EMC virus brought about a decrease in glucose-stimulated protein and insulin biosynthesis as early as 2 h after infection without concomitant effects on insulin release. This inhibition of protein biosynthesis was still apparent at 20 h post-infection, at which time insulin release was found to be markedly elevated, and the islet insulin content was moderately decreased. At 44 h post-infection, glucose-induced insulin biosynthesis was preferentially inhibited. Infected islets at this later time point also displayed elevated levels of insulin release, and a marked loss of islet insulin content. When insulin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were assessed by dot-blot hybridization using appropriate cDNA probes, levels of insulin mRNA were shown to decrease steadily during the first 20 h of infection, in contrast with the levels of GAPDH mRNA. At 44 h post-infection, both types of mRNA were markedly decreased. It is suggested that there is an initial early ‘shut-off’ of protein synthesis without other detectable changes in islet function. This is followed by a phase where both insulin mRNA levels and insulin synthesis are dramatically decreased.

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