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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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Exposure of different RTA residues to the membrane interior. A, the tube worm representation of the α-carbon backbone of the RTA crystal structure is shown. Amino acids that are exposed to the membrane at 20 °C are shown in green, whereas amino acids that are exposed to the nonpolar lipid core only at 37 °C are indicated in red. B, the emission intensities of NBD-labeled RTA mutants (450 nm in buffer H) were measured before and after the addition of PCPS liposomes. Emission intensities of parallel samples containing either 22.5 mol% 12NOPC (F12NO) or 22.5 mol% of PC (F0) were compared at 20 °C (gray bars) or at 37 °C (black bars), respectively. The averages of at least three independent experiments are shown, and the error bars indicate the S.D. of the experiments. Sequence numbers of NBD-labeled amino acids are shown on the x axis. *, p = 0.0004; **, p = 0.04; ***, p = 0.07 when compared with the corresponding quenching efficiency at 20 °C (Student's t test). C and D, the ratio of RTA mutant (450 nm in buffer H) NBD emission intensity (C) and the change in λem max (D) are shown before (F0) and after (FKRM) binding to KRMs (20 eq), either at 20 °C (gray bars) or at 37 °C (black bars). The average of at least three independent experiments is shown, and the error bars indicate the S.D. of the experiments. A, *, p < 0.00004; **, p < 0.0002. B, *, p < 0.00001; **, p < 0.001 when the measurements at 20 °C were compared with those at 37 °C (Student's t test).
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fig8: Exposure of different RTA residues to the membrane interior. A, the tube worm representation of the α-carbon backbone of the RTA crystal structure is shown. Amino acids that are exposed to the membrane at 20 °C are shown in green, whereas amino acids that are exposed to the nonpolar lipid core only at 37 °C are indicated in red. B, the emission intensities of NBD-labeled RTA mutants (450 nm in buffer H) were measured before and after the addition of PCPS liposomes. Emission intensities of parallel samples containing either 22.5 mol% 12NOPC (F12NO) or 22.5 mol% of PC (F0) were compared at 20 °C (gray bars) or at 37 °C (black bars), respectively. The averages of at least three independent experiments are shown, and the error bars indicate the S.D. of the experiments. Sequence numbers of NBD-labeled amino acids are shown on the x axis. *, p = 0.0004; **, p = 0.04; ***, p = 0.07 when compared with the corresponding quenching efficiency at 20 °C (Student's t test). C and D, the ratio of RTA mutant (450 nm in buffer H) NBD emission intensity (C) and the change in λem max (D) are shown before (F0) and after (FKRM) binding to KRMs (20 eq), either at 20 °C (gray bars) or at 37 °C (black bars). The average of at least three independent experiments is shown, and the error bars indicate the S.D. of the experiments. A, *, p < 0.00004; **, p < 0.0002. B, *, p < 0.00001; **, p < 0.001 when the measurements at 20 °C were compared with those at 37 °C (Student's t test).

Mentions: Exposure of Specific RTA Residues to Bilayer Lipids—Above, we have documented a temperature-dependent exposure of RTA259-NBD to the nonpolar lipid core of the membrane that coincides with a conformational change in RTA. Does this conformational change constitute the partial unfolding of RTA that has long been postulated to precede its retrotranslocation through the ER (5, 6)? If so, the unfolding might proceed randomly to create a collection of different partially unfolded RTA molecules or via an ordered pathway of specific structural rearrangements that reproducibly create a specific membrane-exposed topography. We therefore examined the membrane exposure of various sites within RTA (Fig. 8A). Each of these derivatives bound to KRMs as shown by increases in NBD anisotropy upon KRM exposure (supplemental Tables S1 and S2).


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)

Exposure of different RTA residues to the membrane interior. A, the tube worm representation of the α-carbon backbone of the RTA crystal structure is shown. Amino acids that are exposed to the membrane at 20 °C are shown in green, whereas amino acids that are exposed to the nonpolar lipid core only at 37 °C are indicated in red. B, the emission intensities of NBD-labeled RTA mutants (450 nm in buffer H) were measured before and after the addition of PCPS liposomes. Emission intensities of parallel samples containing either 22.5 mol% 12NOPC (F12NO) or 22.5 mol% of PC (F0) were compared at 20 °C (gray bars) or at 37 °C (black bars), respectively. The averages of at least three independent experiments are shown, and the error bars indicate the S.D. of the experiments. Sequence numbers of NBD-labeled amino acids are shown on the x axis. *, p = 0.0004; **, p = 0.04; ***, p = 0.07 when compared with the corresponding quenching efficiency at 20 °C (Student's t test). C and D, the ratio of RTA mutant (450 nm in buffer H) NBD emission intensity (C) and the change in λem max (D) are shown before (F0) and after (FKRM) binding to KRMs (20 eq), either at 20 °C (gray bars) or at 37 °C (black bars). The average of at least three independent experiments is shown, and the error bars indicate the S.D. of the experiments. A, *, p < 0.00004; **, p < 0.0002. B, *, p < 0.00001; **, p < 0.001 when the measurements at 20 °C were compared with those at 37 °C (Student's t test).
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fig8: Exposure of different RTA residues to the membrane interior. A, the tube worm representation of the α-carbon backbone of the RTA crystal structure is shown. Amino acids that are exposed to the membrane at 20 °C are shown in green, whereas amino acids that are exposed to the nonpolar lipid core only at 37 °C are indicated in red. B, the emission intensities of NBD-labeled RTA mutants (450 nm in buffer H) were measured before and after the addition of PCPS liposomes. Emission intensities of parallel samples containing either 22.5 mol% 12NOPC (F12NO) or 22.5 mol% of PC (F0) were compared at 20 °C (gray bars) or at 37 °C (black bars), respectively. The averages of at least three independent experiments are shown, and the error bars indicate the S.D. of the experiments. Sequence numbers of NBD-labeled amino acids are shown on the x axis. *, p = 0.0004; **, p = 0.04; ***, p = 0.07 when compared with the corresponding quenching efficiency at 20 °C (Student's t test). C and D, the ratio of RTA mutant (450 nm in buffer H) NBD emission intensity (C) and the change in λem max (D) are shown before (F0) and after (FKRM) binding to KRMs (20 eq), either at 20 °C (gray bars) or at 37 °C (black bars). The average of at least three independent experiments is shown, and the error bars indicate the S.D. of the experiments. A, *, p < 0.00004; **, p < 0.0002. B, *, p < 0.00001; **, p < 0.001 when the measurements at 20 °C were compared with those at 37 °C (Student's t test).
Mentions: Exposure of Specific RTA Residues to Bilayer Lipids—Above, we have documented a temperature-dependent exposure of RTA259-NBD to the nonpolar lipid core of the membrane that coincides with a conformational change in RTA. Does this conformational change constitute the partial unfolding of RTA that has long been postulated to precede its retrotranslocation through the ER (5, 6)? If so, the unfolding might proceed randomly to create a collection of different partially unfolded RTA molecules or via an ordered pathway of specific structural rearrangements that reproducibly create a specific membrane-exposed topography. We therefore examined the membrane exposure of various sites within RTA (Fig. 8A). Each of these derivatives bound to KRMs as shown by increases in NBD anisotropy upon KRM exposure (supplemental Tables S1 and S2).

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