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Ricin A chain insertion into endoplasmic reticulum membranes is triggered by a temperature increase to 37 {degrees}C.

Mayerhofer PU, Cook JP, Wahlman J, Pinheiro TT, Moore KA, Lord JM, Johnson AE, Roberts LM - J. Biol. Chem. (2009)

Bottom Line: At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer.RTA is then stably bound to the membrane because it is nonextractable with carbonate.Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas 77843-1114, USA.

ABSTRACT
After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 degrees C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 degrees C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.

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Temperature dependence of the RTA259-NBD probe environment. Net emission scans (λex = 468 nm) of RTA259-NBD (450 nm) are shown in the presence of either microsome buffer (A) or of KRMs (B; 15-20 eq) at increasing temperatures in buffer H (A and B: only 4, 20, 30, and 37 °C are shown). The λem max at different temperatures is shown in (C). The averages of three independent experiments are shown, and the error bars indicate the S.D. of the experiments. However, most of the error bars in A and B are smaller than the circles on the graph.
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fig3: Temperature dependence of the RTA259-NBD probe environment. Net emission scans (λex = 468 nm) of RTA259-NBD (450 nm) are shown in the presence of either microsome buffer (A) or of KRMs (B; 15-20 eq) at increasing temperatures in buffer H (A and B: only 4, 20, 30, and 37 °C are shown). The λem max at different temperatures is shown in (C). The averages of three independent experiments are shown, and the error bars indicate the S.D. of the experiments. However, most of the error bars in A and B are smaller than the circles on the graph.

Mentions: The NBD Emission Spectrum of Membrane-bound RTA259-NBD Is Temperature-dependent—To determine at which temperature the NBD environment of membrane-exposed RTA changes, we compared the emission spectra of free RTA with those of membrane-bound RTA as a function of temperature. When incubated without KRMs, the fluorescence intensity of RTA259-NBD declined with increasing temperature (Fig. 3A) because of the reduced quantum yield of the NBD dye at higher temperatures. In the presence of membranes, the same effect was observed until the temperature reached 30 °C. However, when the temperature was further increased to 37 °C, a substantial increase in RTA259-NBD fluorescence intensity was observed, as well as a significant blue shift in λem max (Fig. 3, B and C). Thus, a relatively small increase in temperature from 30 to 37 °C caused a dramatic spectral change and hence a highly temperature-dependent transition from an aqueous to a hydrophobic environment for the NBD dye at the C terminus of RTA. To determine whether this change in RTA environment originated from a phase change in the lipid bilayer, 1,6-diphenyl-1,3,5-hexatriene was incorporated into KRMs to monitor bilayer fluidity. Because no sudden change in the fluidity or phase of the ER lipids occurred between 20 and 37 °C (supplemental Fig. S1), the sharp temperature-dependent transition from an aqueous to a hydrophobic environment for the C terminus of RTA most likely results from a conformational change in the membrane-bound RTA protein that occurs between 30 and 37 °C.


Ricin A chain insertion into endoplasmic reticulum membranes is triggered by a temperature increase to 37 {degrees}C.

Mayerhofer PU, Cook JP, Wahlman J, Pinheiro TT, Moore KA, Lord JM, Johnson AE, Roberts LM - J. Biol. Chem. (2009)

Temperature dependence of the RTA259-NBD probe environment. Net emission scans (λex = 468 nm) of RTA259-NBD (450 nm) are shown in the presence of either microsome buffer (A) or of KRMs (B; 15-20 eq) at increasing temperatures in buffer H (A and B: only 4, 20, 30, and 37 °C are shown). The λem max at different temperatures is shown in (C). The averages of three independent experiments are shown, and the error bars indicate the S.D. of the experiments. However, most of the error bars in A and B are smaller than the circles on the graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665077&req=5

fig3: Temperature dependence of the RTA259-NBD probe environment. Net emission scans (λex = 468 nm) of RTA259-NBD (450 nm) are shown in the presence of either microsome buffer (A) or of KRMs (B; 15-20 eq) at increasing temperatures in buffer H (A and B: only 4, 20, 30, and 37 °C are shown). The λem max at different temperatures is shown in (C). The averages of three independent experiments are shown, and the error bars indicate the S.D. of the experiments. However, most of the error bars in A and B are smaller than the circles on the graph.
Mentions: The NBD Emission Spectrum of Membrane-bound RTA259-NBD Is Temperature-dependent—To determine at which temperature the NBD environment of membrane-exposed RTA changes, we compared the emission spectra of free RTA with those of membrane-bound RTA as a function of temperature. When incubated without KRMs, the fluorescence intensity of RTA259-NBD declined with increasing temperature (Fig. 3A) because of the reduced quantum yield of the NBD dye at higher temperatures. In the presence of membranes, the same effect was observed until the temperature reached 30 °C. However, when the temperature was further increased to 37 °C, a substantial increase in RTA259-NBD fluorescence intensity was observed, as well as a significant blue shift in λem max (Fig. 3, B and C). Thus, a relatively small increase in temperature from 30 to 37 °C caused a dramatic spectral change and hence a highly temperature-dependent transition from an aqueous to a hydrophobic environment for the NBD dye at the C terminus of RTA. To determine whether this change in RTA environment originated from a phase change in the lipid bilayer, 1,6-diphenyl-1,3,5-hexatriene was incorporated into KRMs to monitor bilayer fluidity. Because no sudden change in the fluidity or phase of the ER lipids occurred between 20 and 37 °C (supplemental Fig. S1), the sharp temperature-dependent transition from an aqueous to a hydrophobic environment for the C terminus of RTA most likely results from a conformational change in the membrane-bound RTA protein that occurs between 30 and 37 °C.

Bottom Line: At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer.RTA is then stably bound to the membrane because it is nonextractable with carbonate.Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas 77843-1114, USA.

ABSTRACT
After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 degrees C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 degrees C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.

Show MeSH
Related in: MedlinePlus