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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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Related in: MedlinePlus

RTA259-NBD binds to microsomes differently at 4 °C and 37 °C. Anisotropy measurements (A) and emission scans (B; λex = 468 nm) of RTA259-NBD (450 nm) were performed before (-KRM) and immediately after the addition of ER microsomal membranes (+KRM; 15-20 eq) in buffer H. Emission intensity and anisotropy data were corrected by the subtraction of the signal obtained from an equivalent NBD-free RTA sample. The averages of at least three independent experiments are shown, and the error bars indicate the S.D. of the experiments.
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fig2: RTA259-NBD binds to microsomes differently at 4 °C and 37 °C. Anisotropy measurements (A) and emission scans (B; λex = 468 nm) of RTA259-NBD (450 nm) were performed before (-KRM) and immediately after the addition of ER microsomal membranes (+KRM; 15-20 eq) in buffer H. Emission intensity and anisotropy data were corrected by the subtraction of the signal obtained from an equivalent NBD-free RTA sample. The averages of at least three independent experiments are shown, and the error bars indicate the S.D. of the experiments.

Mentions: To determine whether RTA259-NBD binding to KRMs can be detected spectroscopically, the membrane dependence of both NBD anisotropy and emission intensity was measured. After determining the anisotropy of free RTA259C-NBD, KRMs were added at either 4 °C or 37 °C. The resulting increases in anisotropy (Fig. 2A) showed that the slowly rotating microsomes significantly slowed the rotational rate of the protein and its NBD, thereby demonstrating that RTA259-NBD bound to the KRMs at both 4 and 37 °C. No change in the NBD emission spectrum of RTA259-NBD was observed when KRMs were added to the protein at 4 °C (Fig. 2B). However, upon RTA259-NBD incubation with KRMs at 37 °C, a substantial increase in fluorescence intensity was observed and also a significant blue shift in the wavelength of maximum emission intensity (λem max) (Fig. 2B). Thus, the NBD at residue 259 moved from an aqueous environment to a more hydrophobic milieu when RTA was exposed to microsomal membranes at 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)

RTA259-NBD binds to microsomes differently at 4 °C and 37 °C. Anisotropy measurements (A) and emission scans (B; λex = 468 nm) of RTA259-NBD (450 nm) were performed before (-KRM) and immediately after the addition of ER microsomal membranes (+KRM; 15-20 eq) in buffer H. Emission intensity and anisotropy data were corrected by the subtraction of the signal obtained from an equivalent NBD-free RTA sample. The averages of at least three independent experiments are shown, and the error bars indicate the S.D. of the experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: RTA259-NBD binds to microsomes differently at 4 °C and 37 °C. Anisotropy measurements (A) and emission scans (B; λex = 468 nm) of RTA259-NBD (450 nm) were performed before (-KRM) and immediately after the addition of ER microsomal membranes (+KRM; 15-20 eq) in buffer H. Emission intensity and anisotropy data were corrected by the subtraction of the signal obtained from an equivalent NBD-free RTA sample. The averages of at least three independent experiments are shown, and the error bars indicate the S.D. of the experiments.
Mentions: To determine whether RTA259-NBD binding to KRMs can be detected spectroscopically, the membrane dependence of both NBD anisotropy and emission intensity was measured. After determining the anisotropy of free RTA259C-NBD, KRMs were added at either 4 °C or 37 °C. The resulting increases in anisotropy (Fig. 2A) showed that the slowly rotating microsomes significantly slowed the rotational rate of the protein and its NBD, thereby demonstrating that RTA259-NBD bound to the KRMs at both 4 and 37 °C. No change in the NBD emission spectrum of RTA259-NBD was observed when KRMs were added to the protein at 4 °C (Fig. 2B). However, upon RTA259-NBD incubation with KRMs at 37 °C, a substantial increase in fluorescence intensity was observed and also a significant blue shift in the wavelength of maximum emission intensity (λem max) (Fig. 2B). Thus, the NBD at residue 259 moved from an aqueous environment to a more hydrophobic milieu when RTA was exposed to microsomal membranes at 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